f 1 



SYMBIOTIC NITROGEN FIXATION AS INFLUENCED BY THE 
NITROGEN IN THE SOIL 



BY 

WILLIAM ALBERT ALBRECHT 

A . B. University of Illinois, 1911 

B. S: University of Illinois, 1914 
M. S. University of Illinois, 1915 



THESIS 

Submitted in Partial Fulfillment of the Requirements for the Degree of 

Doctor of Philosophy in Agronomy in the Graduate 

School of the University of Illinois 



1919 



SYMBIOTIC NITROGEN FIXATION AS INFLUENCED BY THE 
NITROGEN IN THE SOIL 



BY 
WILLIAM ALBERT ALBRECHT 

A. B. University of Illinois, 1911 

B. S. University of Illinois, 1914 
M. S. University of Illinois, 1915 



THESIS 



Submitted in Partial Fulfillment of the Requirements for the Degree of 

Doctor of Philosophy in Agronomy in the Graduate 

School of the University of Illinois 



1919 



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CONTENTS 

V Introduction 275 

II. Historical 276 

III. Experimental 283 

Plan of the experiment 284 

Analytical methods 287 

Discussion of crop series and results 288 

Series 1 (soybeans) 288 

Nitrates in soil 290 

Nodule production 291 

Nitrogen balance 292 

Series 2 (cowpeas) 296 

Nodule production 298 

Nitrates in soil 298 

Nitrogen balance 299 

Series 3 (Cowpeas) 302 

Nodule production 303 

Nitrates in soil 304 

Nitrogen balance 305 

Nitrogen changes in soils during growth of legumes 309 

Distribution of nitrogen in tops and roots of cowpeas 311 

IV. Summary 314 

V. Conclusions 316 



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Reprinted from Son. Science, 
Vol. IX, No. 5, May, 1920 



SYMBIOTIC NITROGEN FIXATION AS INFLUENCED BY 
THE NITROGEN IN THE SOIL^ 

WILLIAM ALBERT ALBRECHT 

Formerly Fellow in Agronomy, University of Illinois^ 
Received for publication February 10, 1920 

I. INTRODUCTION 

Of the important elements necessary for growing plants, nitrogen is the one 
that presents our most serious problem and is most apt to be deficient. Early 
studies on the different forms in which nitrogen may serve for plant growth 
seemed to have it definitely settled that only combined nitrogen could be used 
by plants (6, 30). Atwater (2), however, showed that legumes, quite contrary 
to earher beliefs, were able indirectly to utilize the elementary nitrogen. This 
prompted many researches and opened many discussions, which soon estab- 
lished the fact that certain plants, belonging to the Leguminosae, were able to 
obtain the gaseous nitrogen of the air through the action of bacteria living in 
the nodules on their roots. 

When the fact became known that legumes are able to use the nitrogen of 
the air through a mutually beneficial relationship with bacteria, numerous 
studies of these plants were undertaken to determine the manner in which 
they take nitrogen from the air and incorporate or fix it in their tissues. 

This process of "symbiotic nitrogen fixation," as it has been named, has 
taken on considerable significance in the attempt to maintain the nitrogen 
supply for plant growth. The fact that it offers a means of utiHzing the 
unlimited supply of nitrogen of the air in place of the costly nitrogenous 
fertilizers, has served as an incentive to study this process and the factors 
which influence its highest development. Any information giving a clearer 
understanding of the process of symbiotic nitrogen fixation may be justified 
as contributing to the large agricultural problem of maintaining the supply 
of nitrogen in the soil in sufficient amounts to insure maximum crop produc- 
tion. The following research is a contribution to the process of nitrogen 
fixation by legumes as influenced by the amount of nitrogen present in the soil 
in both the organic and inorganic forms. 

1 A thesis submitted to the Faculty of the Graduate School of the University of Illinois 
in partial fulfillment of the requirements for the Degree of Doctor of Philosophy, June, 1919. 

2 Present Associate Professor of Soils, University of Missouri. 

275 

SOIL SCIENCE, VOL. IX. NO. 5 



276 WILLIAM ALBERT ALBRECHT 



II. HISTORICAL 



The literature on the subject of symbiotic nitrogen fixation is rather exten- 
sive and has been well collected in bibliographies by Jacobitz (27), by Burrill 
(7), and others, so that no extensive review on the subject is necessary. Only 
those papers deaUng particularly with this process as influenced ^by the nitro- 
gen content of the soil will be considered. " ' - " ' 

General statements are common in saying that the legume fulfills its needs 
for nitrogen from the soil and later resorts to the supply in the air. Conn (8) 
makes the statement that "legumes appear to prefer taking their nitrogenous 
material directly from the nitrogenous foods in the soil when these are present 
in abundance. But if the soil does not furnish the proper nitrogen, then 
recourse is had to atmospheric nitrogen, through the agency of tubercle 
organisms." Van Slyke (46) ventures a similar opinion in which he says, 
" when supplied with available nitrogen compounds, the bacteria fail to make 
use of atmospheric nitrogen," Hopkins (25, p. 217) agrees in substance with 
this. "Clover and other legumes," he says, "take available nitrogen from 
the soil in preference to the fixation of free nitrogen from the air, the latter 
being drawn upon only to supplement the soil's supply and thus balance the 
plant-food ration." 

Early works show that nodules are present when the bacteria become estab- 
lished and when the plant uses atmospheric nitrogen. In much of the litera- 
ture the nodules represent nitrogen fixation and unless this modification of 
the root is present, no use of gaseous nitrogen is believed to be taking place. 
The importance of nodules in nitrogen fixation was established early by 
Hellriegel and Wilfarth (19) as one of the fundamental facts when they say, 
"the nodules of the roots must not be considered as simply reservoirs of 
albuminoid substances; their relation to the assimilation of free nitrogen is 
that of cause to effect." In most works cited in the following discussion, the 
effects of the nitrogen in the soil as a cultural medium, are reported as favorable 
or unfavorable to the nodule production, and hence to the nitrogen fixation. 

Moore (34) apparently does not agree with this general conception of the 
importance of nodules. He believes that it is possible for the bacteria to 
enter the roots and be of benefit without evincing their presence by such 
external evidence as nodules. He fails to beheve that even if the absence of 
nodules might permit some nitrogen fixation, this would not prohibit the same 
performance in their presence. 

Rautenberg and Kuhn (43) ventured perhaps the earliest statement con- 
cerning the relation of nitrogen fixation to the nitrogen in the medium for 
plant growth. In their work Viciafaba, growing in a nitrogen-free solution, 
developed numerous nodules, while in the presence of nitrates no nodules 
appeared. De Vries (10) obtained similar results while studying nodules as 
a storage for nitrogen. In the absence of nitrogen from the cultural solution, 
many nodules of normal structure were produced, but otherwise scarcely 
any developed. 



SYMBIOTIC NITROGEN FIXATION 277 

Schindler (44) working with clovers, vetch, serradella, kidney vetch, and 
beans in water cultures concludes that, in general, solutions rich in nitrogen 
are less disposed toward giving nodules than those lacking in this element. 
These same plants grown in soil fertihzed with compost or manure as com- 
pared with soils low in nitrogen gave larger and more numerous nodules in 
every case with the latter soil. Tschirch (45) in studying nodules, as a means 
of nitrogen storage, says that it is established that nodules grow more profusely 
in soils poor in nitrogen than in those rich in humus. 

Vines (47) treated soils with 1 per cent potassium nitrate and found nodular 
development decreased, and also an indication that as the amount of nitrate 
diminished, the development of nodules became more marked. Similar 
depressive effects on nodule production by nitrates are reported also by 
Baszler (3) and by Laurent (28). 

These studies on the influence of the nitrogen content of the soil on the 
nodule development, take this development as a measure of nitrogen fixation, 
but fail, however, to substantiate their contentions with careful chemical 
analyses to show that the total nitrogen in the plant and soil has truly increased. 

Frank (13) studied conditions of the plant as influenced by inoculation and 
other factors. In using soils rich in humus as compared with those very poor 
in this respect, he was led to believe that when humus is present in sufficient 
amounts the bacteria are dispensable and serve with no benefit to the plant. 
Where humus is lacking the bacteria are active. This explains why legumes 
can be grown on sand when all minerals are supplied even when no humus 
is present. 

Atwater (2) in growing peas in sand supplied with nutrient solutions con- 
taining varying amounts of potassium and calcium nitrates, found nitrogen 
fixation taking place. He measured it by the increase in the total nitrogen 
present at the close of the experiment as compared with that at the start. 
Table 1 is taken from his data and shows fixation when a large nitrogen ration 
is supplied. 

Woods (50) (of Connecticut) grew scarlet clover in sand with a nutritive 
solution and accounted for all the nitrogen in the experiment, as did Atwater, 
by analysis of material at the outset and at the close. With no nitrogen in 
the solution, 18 plants fixed an average of 37 mgm. per plant. When 40 mgm. 
of nitrogen as calcium nitrate and potassium nitrate were added at the start, 
the fixation was reduced to 30 mgm. per plant. Vetch responded differently, 
fixing 20 mgm. in the former case and 47 in the latter. Cowpeas treated with 
nitrate fixed amounts varying from 87 to 129 mgm. of nitrogen. His data 
show a larger part of the nitrogen in the roots when nitrate was omitted than 
when it was added. He says, "all the plants grown without added nitrogen 
gained in nitrogen. Some of the plants supplied with nitrate showed a loss. 
The gain has occurred where root nodules are developed and without them 
there was no gain of any account." 

Prazmowski (42) worked on an experiment similar to that of Woods to find 
out if the nitrates in the soil hindered or aided the bacteria in entering the 



278 



WILLIAM ALBERT ALBRECHT 



plant and fixing nitrogen. Peas in sterile sand cultures containing 300 mgm, 
of nitrogen fixed 50 mgm. With no nitrogen in the sand and only 12 mgm. 
in the seed, 70 mgm. were taken from the air. In working with water cul- 
tures, nitrates held down nitrogen fixation and nodule production. In the 
absence of nitrates the fixation varied from 17 to 83 mgm. per plant. He 
says that the nitrogen content of the soil influences the time at which the 
nodule empties itself. The presence of nitrogen in the soil brings this change 
at the time of seed formation, but its absence from the soil permits the pro- 
cess of nodule growth and nitrogen fixation to go on slowly during the life of 
the plant with an increase in it at the time of seed formation. 



TABLE 1 



Nitrogen fixed by peas grown in nutrient solutions containing nitrates 
(Taken from Atwater) 





NUMBER 
OF 


NITROGEN SUPPLIED 


NITROGEN AT CLOSE 


GAIN OR 




EXPERI- 
MENT 


In seeds 


In 
solution 


Total 


Vines, 
roots, etc. 


Residual 
solution 


Total 


NITROGEN 






mgm. 


mgm. 


mgrn. 


mgm. 


mgm. 


mgm. 


mgm. 




1 


36.7 


59.4 


96.1 


116.4 


1.4 


117.8 


-21.7 




3 


72.6 


59.4 


132.0 


158.9 


3.8 


162.7 


-30.7 


Group I. Small ni- 


5 


34.2 


59.4 


93.6 


156.1 


0.0 


156.1 


-62.5 


trogen ration 


7 


71.5 


59.4 


130.9 


158.1 


0.0 


158.1 


-27.2 




9 


35.3 


59.4 


94.7 


186.5 


1.4 


187.9 


-93.2 




11 


72.5 


59.4 


131.9 


210.9 


2.7 


213.6 


-81.7 




2 


34.4 


136.9 


171.3 


178.9 


2.0 


180.9 


-9.6 




4 


75.2 


136.9 


212.1 


200.6 


12.8 


213.4 


-1.3 


Group II. Large ni- 


6 


34.8 


136.9 


171.7 


149.6 


1.2 


150.8 


-20.9 


trogen ration 


8 


70.3 


136.9 


207.2 


197.5 


12.7 


210.2 


-3.0 




10 


34.6 


136.9 


171.5 


277.8 


35.7 


313.5 


-142.0 




12 


68.8 


136.9 


205.7 


260.2 


45.7 


305.9 


-100.2 



This work cited last, gives a different degree of effect for nitrates in a sand 
or open medium than it does for nitrates in water cultures. This fact may 
be of significance in explaining the injurious effects on nodule growth and 
nitrogen fixation which are attributed in many cases to the nitrates. The 
use of solutions may be inadequate for an experiment of this nature. 

To test the effects of different forms of nitrogen on the nodule growth of 
legumes, Frank (14) used calcium nitrate, ammonium sulfate and urea. He 
analyzed seeds at the start and total plants at the close, and found that for 
the lupine the greatest growth and nitrogen increase in the plant, as well as 
the most profuse nodule production, took place in the absence of all nitro- 
genous compounds. The pea behaved similarly. He measured the nitrogen 
fixed in sand and soil by lupines, peas and red clover, obtaining the results 
given in table 2. 



SYMBIOTIC NITROGEN FIXATION 



279 



Frank's data show that the yellow lupine fixes less nitrogen on a humus 
soil than on a sand soil, or that soils with a higher nitrogen content fix less 
nitrogen with this legume. It may be possible that physical differences or 
other factors were responsible, for he gives no detailed description of relations 
other than nitrogen in the soil. 

For the peas and clover the case is different. Both gave a decided increase 
in the soil rich in nitrogen. For the lupine he beheves that its nitrogen- 
fixing power is less in a nitrogen-rich soil than in one very poor in this respect. 
Nevertheless, a soil already rich in nitrogen may be enriched in this element 



TABLE 2 



Nitrogen fixed in sand and soil by lupines, peas and red clover 
(Taken from Frank) 



KIND or son. 



DRY 
WEIGHT 

HAR- 
VESTED 



In seed 
and in- 
oculum 



In 

harvest 



IN- 
CREASE 



NITROGEN IN SOIL 



Outset Close 



INFECTION 



Lupine 



Sand 

Humus soil 

Humus soil 

Sand 

Sterilized sand not in 

oculated 

Humus soil 





gm. gm. 


gm. 




per cent per cent 


5 


14.760.035 


0.3609 


10.3 


0.0096 0.0157 


4 


23.32 0.0364 


0.2816 


7.7 


0.1076 0.1208 



1 large nodule 
1 to 7 nodules 



Peas 



37.98 



0.0282 



0.7467 



26.5 



0.1076 



0.1253 



Of bean size 



Red clover 





44.33 


0.0457 


0.7087 


15.5 


0.0073 


0.0105 




7.18 


0.0457 


0.0687 


1.5 


0.0073 


0.0079 




222.02 


0.0457 


4.6406 


105.5 


0.1076 


0.1184 



Rich in nodules 

Few nodules 
Few nodules 



by means of legumes. Peas and clover, he believes, reach their maximum 
fixing capacity only when using nitrogenous substances, especially nitrates, 
to supplement the bacteria on their roots, even though the direct opposite is 
true for the yellow lupine. The nitrogen enriching effect of legumes takes 
place not only in soils poor in nitrogen, but also in the better soils, rich in 
humus. 

The above conclusion is quite the opposite to that of Maercker (31), who 
used the yellow lupine in sterilized and inoculated sand with varying amounts 
of potassium nitrate added. He found in this experiment that nitrates did 
not hinder or lessen the abihty of the lupine as a nitrogen fixer. Nobbe and 
Hiltner (35) reported the diameter of Robinia nodules as 8 mm. in nitrogen-free 
soil, and 0.5 mm. in soil treated with nitrates. 



280 WILLIAM ALBERT ALBRECHT 

Perhaps the most careful work in the early study of nitrogen fixation is 
that of Aeby (1) in his attempt to see if non-legumes would give nitrogen 
fixation. He used two soils, one a clay soil with 0.0783 per cent nitrogen and 
the other a "humus-rich" soil with 0.4050 per cent nitrogen. They were 
used in growing peas without nitrogen treatment, and with nitrogen added 
at four intervals to make a total of 2 gm. per pot of 4 kgm. of soil. Analyses 
were made for total nitrogen in all materials at the beginning and at the close. 
Any increase present at the close over that at the beginning represented fixa- 
tion of nitrogen. This method of analysis gave a loss of nitrogen in fallow 
pots but a decided increase for those on which peas were grown. Peas grow- 
ing in a rich soil fixed 1.976 gm. nitrogen per pot, while in the same soil treated 
with nitrogen, the fixation was but 1.621 gm. In the clay soil the corre- 
sponding figures are 2.759 gm. and 1.987 gm., respectively; a decrease of 
0.355 and 0.772 gm. due to the nitrogen added. This indicates that in the 
soil rich, as well as one poor in nitrogen the addition of nitrogen depressed 
the fixation of atmospheric nitrogen. Furthermore, in the soil which was 
low in nitrogen the amount taken from the air by the peas was greater than 
in the rich soil, both when untreated and treated with nitrogen. Accordingly 
there was less fixation with increased amounts of nitrogen in the soils, which 
agrees with some of the preceding works cited. In the soil left fallow, he 
failed to recover as much nitrogen at the close as was present at the beginning. 
In the distribution of nitrogen in the roots and tops of plants, his results agree 
with those of Woods. 

Nobbe and Hiltner (36) go farther in their statements than many others 
and conclude from a study of cross inoculation, that nodules have no influence 
on plant growth when plenty of soil nitrogen is available. 

Salts containing nitrogen were used by Marchall (32) and found to inhibit 
nodule production in the following concentrations: alkaline nitrates 1 part 
in 10,000 and ammonium salts 1 part in 2000. By using soybeans and meas- 
uring the nitrogen increase in t^rms of the crop, when sterile or inoculated, or 
treated with nitrate nitrogen, Nobbe and Richter (38) found that with 
increased amounts of soluble nitrogen or humus substance added, the total 
nitrogen content of the crop decreased. They beHeved that small amounts 
of soluble nitrogen are beneficial to the young plants — at least until bacteroids 
are formed. According to them, inoculation was best in the absence of 
nitrates and decreased with the increase of the latter. 

To judge the value of legumes as nitrogen fixers, Wohltmann and Bergene 
(51) used a variety of soils ranging in nitrogen from 0.046 per cent to 0.205 per 
cent and a peat soil with 1.650 per cent. These soils were treated with either 
ammonium nitrate or ammonium sulfate and planted to a number of legumes. 
They judged the amount of nitrogen taken from the air in terms of the number 
of nodules produced, and give the following results and conclusions in regard 
to the influence of the nitrogen in the soil on the amount of nitrogen fixed. 
On all soils to which ammoniimi nitrate was added the nodules failed to 



SYMBIOTIC MTROGElv nXATION 



281 



develop and the plants grew weU in tke absence of tliem, Anamonimn sulfate 
suppressed nodtde production completely in nine cases and almost completely 

in two cases. 

These men belie\^e that legumes do not need the help of baaeria and atmos- 
pheric nitrogen when there is present in the soil an abimdant supply of avail- 
able nitrogen, and that the>^ use soil nitrogen ahnost exclusively. According 
to these conations, legum^ used as green manure would not add nitrogen to 
that soil whose a\^ailable supply of this essential element is high. The}' point 
out that it is the "a^^ailable" nitrogen rather than the "total" nitrogen that 
has a detrimental influence on nitrogen fixation. 

Similar resnlts were obtained about 1904 by Kobbe and Richter (39) who 
undertook to determine the efiect of soluble nitrogen in soil on the amomit 
of nitrogen taken from the air by vetch. The)^ measured fixation by differ- 
ence between total nitrogen in plants that were inoculated and those that were 
not so treated. The increase in the nitrogen content caused by bacteria was 

TABLES 
mtrogen in »stc*, inocuLaied wni urdnocidaUd, IreateA iviih nUraie niirogen 

'Trom Nob>>; arid Eichtsr) 



ys irrnfjszsi ' 500 maL. \ 1000 jsgil 

ADKEI/ ^STTEOGZy ADHED 3S3IE.OGEJJ tJJOZSi 



T-,— vv^r^ V-. 1.533 



No: 'viArjoi^v&i (gm.) 

X/ir erence ' gm. ) 

Increase f^per cait of t<itzS). 



0.095 

1.438 
93.8 



1.887 
0.389 
1.498 
79-38 



2.295 
0.618 
1.677 
73.07 



Nitrosen ratio - . . - 



considered as coming from the air. Increaang ai^ounts of potassium nitrate 
were added to the soil and th. entire plants analyzed. Tney found the 
results given in table 3. 

According to their figures the effect of inoculation decreases with the mcrease 
in soluble sdl nitrogen. Viliere no soluble nitrogen was added the bacteria 
increased the nitrogen in the plant sixteen-f old. As much as 93 per cent of 
the nitrogen in the inoculated legume came from the atmosphere. ^ ith the 
addition of 500 mgm. of soh-ble nitrogen the increase by baxrteria was ahnost 
four-fold, or about 80 per cent of the nitrogen in the entire plant came from 
the air When more nitrate was added, the depressive effect increased, 
though not proportionaDy. These amounts of soluble nitrogen, according: 
to their data, decreased the action of the baaeria in suppl^-ing the plant ycixsy 

atmospheric nitrogen. -, . in j n 7^ 

\ large A-ariet\- of nitrogenous compounds were tested b>- liamana uz; 
lot thai hrauQii cm nodule piodscdaon witi peas, beans and vetch. He 
found tiaat potasaom nitrate pMisHated nodule formation when used m 
amounts as low as 1 part in 10,000; sodium nitrate required >^ut 1 part m 



282 



WILLIAM ALBERT ALBRECHT 



2000, or the equivalent of 1000 pounds per acre. Urea, oxamide, and 
potassium cyanide were very prejudicial to the production of nodules even 
when used in very dilute solution. Ammonium salts, either as nitrate or sul- 
fate, prohibited nodules on vetch. Table 4 gives the proportions in which 
these compounds were fatal to nodule production. 

This work was done in water cultures and as in previous works cited may 
have given more significant effects than would be true of soils. 

The contention that assimilable nitrogen hinders nodule production is 
further supported by A, Hercke (20) who concludes that, "when the soil 
contains sufficient assimilable nitrogen, the presence of nodules on the roots 
has no influence on the nitrogen content of lupines. When soil is poor in 
nitrogen the presence of nodules increases the absolute as well as the per- 
centage of nitrogen content of the plant." This is contradicted by S. Hercke 
(21) who found that nitrogen compounds as ammoniimi sulfate, potassium 
nitrate and asparagin favored the growth of nodule bacteria. 



TABLE 4 

Nitrogen concentrations fatal to nodule production 
(From Flamand) 



Potassium nitrate . , 

Sodium nitrate 

Calcium nitrate. . . . 
Ammonium nitrate 
Ammonium sulfate 



Pisum sativum 



1:10,000 
1:10,000 
1: 2,000 
1:10,000 
1:10,000 



Vicia 
narbonensis 



1:10,000 
1: 2,000 
1:10,000 
1 : 20,000 
1:20,000 



Faba equina 



1:10,000 
1: 2,000 
1:20,000 
1: 2,000 
1:10,000 



Fred and Graul (17) tested the effect of soluble nitrogenous compounds 
on nitrogen fixation in different kinds of soil and with different kinds of plants. 
In general, they found that increasing amounts of soluble nitrogen depressed 
nodule production, but for this effect a larger amount of soluble nitrogen was 
necessary than would probably ever occur under field conditions. They are 
supported in this by the field experiments of Ewart (11). 

When it was found that bacteria are responsible for nitrogen fixation in 
conjunction with the plant, the question arose concerning their behavior 
in this respect independent of a host. Several researches have been carried 
out to see how nitrogen in various forms in the media influences the nitrogen- 
assimilating capacity of this organism when separated from the plant. In 
1891 Beijerinck (4) found nitrogen fixation taking place by these bacteria 
(since named Ps. radicicola) independent of a plant, when the media contained 
ammonium, sodium, and potassium nitrates. Other early workers who 
reported fixation independent of the plant in the presence of nitrogenous 
compounds were Prazmowski (42), Berthelot (5), and Frank (14), but they 
said it was too small to be significant. Larger nitrogen fixation was reported 



SYMBIOTIC NITROGEN FIXATION 283 

for the organism living independently by Maze (33) and by Lewis and Nichol- 
son (29), the former finding as much as 23 mgm. of nitrogen fixed in 100 cc. 
of medium in 16 days. 

The presence of nitrogen is considered injurious to the bacteria themselves 
according to Moore (34). He beUeves that, "the cultivation of bacteria 
upon media containing appreciable quantities of nitrogen for any length of 
time is sufficient to cause them to lose both the power of infection and that 
of fixing atmospheric nitrogen." This is strongly refuted, however, by Burrill 
and Hansen (7) who grew Ps. radicicola on nitrogen media for 30 months 
without a loss of its special adaptations or its capacity for producing nodules. 

Very recently Fred (15) found that Ps. radicicola fixed more nitrogen when 
a trace of this element was present in the medium, but larger amounts of it 
retarded the process. He is supported by still more recent work of Hills (23) 
who found that fixation independent of the plant was increased by nitrate 
nitrogen. His results vary somewhat, and the fixation is large enough to be 
significant, which may not be wholly true of Fred's results. 

This review of literature indicates that on the fixation of nitrogen by 
legumes, one may expect a significant influence from the nitrogen content of 
the soil. According to several researches soluble or assimilable nitrogen has 
a depressing influence, especially in water cultures and in soils when used 
in larger amounts. Nobbe and Hiltner (37) showed that the water-culture 
method itself was unfavorable for the development of nodules, and conse- 
quently nitrogen additions under such conditions may have been wrongly 
interpreted. The use of soil seems to be the best procedure and duplicates 
most nearly the field relations. Little has been done, however, in using 
carefully analyzed soil to measure the influence of soil nitrogen on the process 
of nitrogen fixation under those conditions generally prevailing in the soil. 
The opinions on this question are varied, though the more recent ones em- 
phasize the assimilable or soluble nitrogen of the soil as being depressive to 
the efficiency of nitrogen-fixing bacteria. In most experiments cited the 
soluble nitrogen was applied as ammonia or nitrate salts and no account was 
taken of the nitrogen in organic matter. Such works fail to settle the signifi- 
cance of the organic matter in the soil concerning symbiotic nitrogen fixation. 

ni. EXPERIMENTAL 

The following experimental work was undertaken in order to study further 
the relation of symbiotic nitrogen fixation to the nitrogen content of soils, 
especially the total nitrogen as obtained by usual soil analysis. In advising 
the use of legumes in a rotation cropping system, especially for the soils of the 
Corn Belt, which are moderately high in total nitrogen, the following questions 
often arise: Will the legumes fix any atmospheric nitrogen in a soil already con- 
taining large amounts of this element? If they do, how efiicient will they be, 
and what significance does the nitrogen content of the soil have on the process? 
The following experimental work was undertaken with the hope of contributing 
a possible answer to these questions. 



284 WILLIAM ALBERT ALBRECHT 

Plan of the experiment 

In experimental work necessitating the measurement of nitrogen fixation, 
two general methods have been widely accepted. The first one consists in 
growing one set of legume plants without bacteria on the roots, and another 
set under Uke conditions but with the organism applied. The difference in 
the nitrogen content of the two sets of plants represents the increase due to 
bacteria. The second method consists of careful analyses of all materials to 
determine the total nitrogen present both at the outset and at the close of the 
experiment. Under carefully controlled conditions, with no nitrogen added, 
the increase of this element at the close over that at the beginning represents 
nitrogen drawn from the atmosphere. 

Objections have been made to the first method on the ground that the soil 
or medium in which the plant grows is not taken into consideration. An in- 
crease in the nitrogen content of the plant because of the bacteria may mean 
that the plant was able to take more nitrogen from the soil and does not prove 
that all such increase in nitrogen came from the air. 

The second method is more detailed and painstaking and gives difficulty 
because of lack of refined methods for determining total nitrogen. However, 
it measures the total nitrogen in all materials concerned at the beginning, 
and again at the close, so that the increase must come from some other source 
than the soil. In carefully controlled conditions this source must be the at- 
mosphere. The latter method was used in this study. 

The soil used for the pot cultures was a yellow silt loam from the unglaci- 
ated area of southern Illinois and contained 625 pounds of nitrogen per 2,000,- 
000 pounds of surface soil. It was extremely low in organic matter, hence in 
such a poor physical condition that at first sight it would appear much like a 
clay or clay loam. It gave an acid reaction, and in order to neutralize this 
it was treated with calcium carbonate at the rate of 2 tons per acre. Plant 
nutrient elements other than nitrogen were added in soluble form during the 
time the plants were growing to assure good fertility, except for nitrogen which 
is the main deficiency in the soil (26). This soil was chosen because it was so 
low in nitrogen, and would offer a low nitrogen basis, which had become stable 
because of years of weathering, and which could be increased by several large 
increments without surpassing the nitrogen content of the more fertile soils. 

Two kinds of legumes were grown, soybeans and cowpeas. The first series 
included soybeans but failed to do as well as expected in greenhouse work. 
They were replaced by cowpeas for the later series which were grown out-of- 
doors during most of the time. Insects molested the plants in all series and 
were combatted with chemicals containing no nitrogen. Red spiders were 
especially prevalent and were sprayed with cold water or potassium sulfide 
solution. During the first series a fungus gnat (Sciaria mycetophilidae) (18) 
infested the soils which were high in organic matter. Later troubles from this 
insect were not experienced. Insect infestations were prevalent on all plants, 



SYMBIOTIC NITROGEN FIXATION 



285 



SO that even though they may have hindered growth to some extent, their 
disturbances would not prevent comparable results. 

In the first series which contained soybeans, four duplicate series of 1 -gallon 
pots were used with the soil treatments given in table 5. 



TABLE s 



Soil treatments in crop series 1 
(Soybeans) 



NITROGEN ADDED TO SOU (POUNDS PER 2,000,000 POUNDS OF SOU,) 



Pot series 1 



1-2 
3-4 
5-6 
7-8 
9-10 



None (not inoculated) 

None 
10 pounds as nitrate 
50 pounds as nitrate 

150 pounds as nitrate 



Pot series 2 



11-12 
13-14 
15-16 
17-18 
19-20 



1000 pounds as clover tops 
2000 pounds as clover tops 
3000 pounds as clover tops 
4000 pounds as clover tops 
5000 pounds as clover tops 



Pot series 3 



21-22 
23-24 
25-26 
27-28 
29-30 



1000 pounds as clover tops and 10 pounds as nitrate 
2000 pounds as clover tops and 10 pounds as nitrate 
3000 pounds as clover tops and 10 pounds as nitrate 
4000 pounds as clover tops and 10 pounds as nitrate 
5000 pounds as clover tops and 10 pounds as nitrate 



Pot series 4 



31-32 
33-34 
35-36 
37-38 
39-40 



1000 pounds as clover tops and 50 pounds as nitrate 
2000 pounds as clover tops and 50 pounds as nitrate 
3000 pounds as clover tops and 50 pounds as nitrate 
4000 pounds as clover tops and 50 pounds as nitrate 
5000 pounds as clover tops and 50 pounds as nitrate 



The addition of nitrate nitrogen and clover tops was made on the basis of 
one acre, or 2,000,000 pounds of soil. In all cases a constant amount of soil 
was used, and the increasing amounts of clover tops added gave increasing 
amounts of substrate for the plants in the series. This treatment also brought 
about decided changes in the soil's physical condition. Four pots were used 
as checks. All pots in the series were inoculated except two of the check pots, 
no. 3 and 4, which remained uncontaminated and showed no nodules at the 
close of the experiment. 



286 



WILLIAM ALBERT ALBRECHT 



TABLE 6 

Soil treatments in crop series 2 
(Cowpeas) 



NITROGEN ADDED TO SOIL (POUNDS PER 2,000,000 POUNDS OF SOIL) 



Pot series 1 



la-2a 
3a-4a 
5a-6a 
7a-8a 
9a-10a 



None 
10 pounds as nitrate 
25 pounds as nitrate 
50 pounds as nitrate 

100 pounds as nitrate 



Pot series 2 



1-2 
3-4 
11-12 
13-14 
15-16 
17-18 
19-20 



None (not inoculated) 

None 

1000 pounds as clover tops 

2000 pounds as clover tops 

3000 pounds as clover tops 

4000 pounds as clover tops 

5000 pounds as clover tops 



TABLE 7 

Soil treatments in crop series 3 
(Cowpeas) 



NITROGEN ADDED TO SOIL (POUNDS PER 2,000,000 POUNDS OF SOIL) 





Pot series 1 


lb-2b 


None 


3b-4b 


50 pounds as nitrates 


5b-6b 


100 pounds as nitrates 


7b-8b 


150 pounds as nitrates 


9b-10b 


200 pounds as nitrates 


llb-12b 


250 pounds as nitrates 


13b-14b 


50 pounds as nitrates (added at intervals) 





Pot series 2 


1-2 


None 


3-4 


None 


11-12 


1000 pounds as clover tops 


13-14 


2000 pounds as clover tops 


15-16 


3000 pounds as clover tops 


17-18 


4000 pounds as clover tops 


19-20 


5000 pounds as clover tops 



SYMBIOTIC NITROGEN FIXATION 287 

The second series was seeded mth cowpeas, and contained two pot series, 
one treated with nitrates, and one with organic matter. Original soil, the 
same as used previously, was treated with nitrate nitrogen, while the series 
treated with organic matter consisted of a part of those soils used in the first 
crop series The four check pots and the ten pots that had been treated with 
nitrogen as clover tops only, were taken to complete this series. These soils 
were in better physical condition and the stage of most rapid decay seemed to 
have been passed. Constant amounts of soil (3300 gm. water-free soil) were 
weighed into each pot. All pots were inoculated except the two check pots, 
but e\ddently these were already inoculated, or became contaminated, for 
nodules were present at the close of the series. This crop series with its treat- 
ment is summarized in table 6. 

The third series was similar in soil treatment to that of the second. Some 
of the original soil was treated ^iih nitrates for one pot series, while those 
soils which had been used in both pre\dous series were used for the other pot 
series. Cowpeas were grown as the crop. The treatments are given in table 
7. 

The last two series in which cowpeas were grown were kept out-of-doors 
near the greenhouse for much of the time. This seemed to favor better growth, 
and lessened the insect troubles. Care was taken to move the plants inside 
before rain and only once did rain fall on each series. It was only a slight 
shower and the effects in adding nitrogen were negligible. 

Analytical methods 

The usual analytical methods were employed. To measure the total nitro- 
gen in organic matter such as seeds and plants, the materials were digested 
with mercury and sulfuric acid or with acid and sodiimi sulfate, neutralized 
with sodium hydroxide and potassium sulfide, distilled into standard acid and 
titrated with standard alkali. For the total nitrogen determination on soils, 
10-gm. samples were first dried for 8 hours at a temperature of 107°-108°C., 
the loss in moisture determined, and then transferred for digestion. Sulfuric 
acid containing salicylic acid was added and allowed to stand for some time, 
usually over night. Sodiimi thiosulfate w^as introduced and heated slowly 
untn frothing ceased. Mercury was then added and digested to clearness, 
potassiimi permanganate being used to assure complete oxidation. This was 
then neutralized with alkali and distilled into standard acid, according to the 
usual procedure. 

For the determination of nitrate nitrogen, samples of soil were dried at 107°- 
108°C. for 8 hours and then extracted with 0.0625 .V hydrochloric acid. After 
being made alkaline, the ammonia was boiled off, and the original volume re- 
placed by nitrogen-free water. Devarda's metal was added, the reduced 
nitrogen distilled into 0.0357 T standard sulfuric acid and titrated. 



288 WILLIAM ALBERT ALBRECHT 

In all the determinations the greatest care was exercised. The organic 
matter was dried, weighed, thoroughly ground, and kept in air-tight containers 
to prevent decided variations in moisture content before an entire series could 
be analyzed. Purest chemicals were used and all reagents were made up in 
large amounts to insure greater uniformity in results. Standard acid for ni- 
trogen in soil was of 1/14 normaUty and with sodium alizarin sulfonate as an 
indicator, titrations checked within 0.2 cc, a variation equivalent to 0.2 mgm. 
nitrogen in 10-gm. samples, or 40 pounds of nitrogen in 2,000,000 pounds of 
soil. For nitrate determinations, a sulfuric acid of 1/28 normality was used 
and 0.2 cc. allowed as variation in duplicates. Determinations in moisture 
loss for 10-gm. samples were usually less than 6 mgm. All calculations and 
determinations for soil were on the water-free basis to insure uniformity. 
Samples were run in triplicate and quadruplicate to offset errors. Only doubly 
distilled nitrogen-free water was used, both in analytical work and in the pot 
cultures. 

Discussion of crop series 

Series 1 (Soybeans) 

The soil for this series was thoroughly mixed in a quantity large enough for 
all the pots. To determine the nitrogen in it, a large sample was taken and 
ground to pass a 100-mesh sieve and then analyzed for total nitrogen. Ac- 
cording to the analysis, 30-gm. quantities of air-dry soil, which were equivalent 
to 29.1970 gm. water-free soil, contained 9.1568 mgm., or 0.03136 per cent 
nitrogen on the latter basis, as an average of five determinations varying less 
than 0.2 mgm. Each pot received 3500 gm. of soil with a moisture content of 
14.01 per cent, or the equivalent of 3421 gm. of water-free soil, and a nit ogen 
content according to the above analysis of 1072 mgm. 

For one pot series the soil was treated with nitrate nitrogen only, at the rate 
of 17.2, 86.0 and 258 mgm. of nitrogen per pot, or the equivalent of 10, 50 and 
150 pounds per 2,000,000 pounds of soil. For three additional pot series the 
nitrogen in the soil was increased at increments equivalent to 1,000 pounds of 
nitrogen per 2,000,000 pounds of soil by means of finely ground clover tops. 
The clover tops contained 2.709 per cent of nitrogen, as an average of 12 analy- 
ses. With 3500 gm. of soil per pot an increment of 1000 pounds per 2,000,000 
— one part in 2000 — required 1.75 gm. of nitrogen, or 64.6 gm. of clover tops. 
Accordingly, for the second pot series enough clover tops were added to give a 
series of the original soil, whose nitrogen was increased at the rate of 1000, 
2000, 3000, 4000 and 5000 pounds per acre. The third series was similar to 
the second except that in addition to the organic matter sodium nitrate also 
was added to all the pots at the rate of 10 pounds of nitrogen per 2,000,000 
pounds of soil, while the fourth series differed from the third only by the addi- 
tion of sodium nitrate at the rate of 50 pounds of nitrogen instead of 10. This 
gave one pot series whose nitrogen was increased as nitrate, one with increas- 



SYMBIOTIC NITROGEN FIXATION 289 

ing nitrogen in organic matter as clover tops, another increasing with clover 
tops and 10 pounds of nitrate nitrogen and the fourth whose nitrogen increased 
as clover tops and 50 pounds of nitrate nitrogen. The addition of varying 
amounts of clover tops to 3500 gm. of soil increased the amount of soil in the 
pots, so that the weight of soil in the different pots was not a constant figure. 

According to Hilgard (22) and others, the best moisture content of soils 
for proper plant development is one-haK of the moisture-holding capacity. 
On this basis, determinations of moisture-holding capacity were made on 
these soil series as modified by additions of clover tops, and the water applied 
to the pots in quantities to give the equivalent of 50 per cent of the 
moisture-holding capacity. An attempt was made to maintain such conditions 
throughout the experiment by weighing the pots at intervals, but the labor in- 
volved was large and the growth of the crops prohibited absolute accuracy. 
It was therefore necessary to apply the water according to one's best judg- 
ment. The presence of large amounts of organic matter caused trouble in 
keeping the moisture at optimum amounts. 

Soybean seeds were selected so that each seed weighed 140 mgm. Six of 
these were planted in each pot, in order that five vigorous plants might be 
assured in spite of low vitality and poor germination. The sixth plant or seed 
was removed after sufficient time had elapsed for the plants to get well started. 
According to the analyses of the beans, each seed contained an average of 9 
mgm. of nitrogen. 

The growth of this series was fair. Numerous replantings were necessary 
because the large amounts of fresh organic matter interfered with proper germi- 
nation of the seeds, a difiiculty experienced also by Fred (16) at Wisconsin and 
by others at Illinois with oats, cowpeas and clover after turning under alfalfa. 
The injurious effects were more marked with the increase in organic matter 
so that on the pots receiving clover tops at the rate of 5000 pounds of nitrogen 
per 2,000,000 pounds of soil, only small plants were produced. These effects 
were not offset by the nitrate nitrogen added at the rate of 10 pounds and 50 
pounds per 2,000,000 along with the organic matter. Some plants "damped- 
off" at the age of four weeks, while others died later. The growth was much 
influenced by the organic matter in the soil, being the best of the entire series 
where 1000 and 2000 pounds of nitrogen had been added as clover tops, but 
decidedly poorer in soil with larger amounts of this material added. The 
plants showed a less deep color, smaller leaves, less branching and more trans- 
location, with the increase in organic matter above the equivalent of 2000 
pounds of nitrogen per acre. 

The effect of the nitrates was marked in the soil when these were added 
alone, but gave no appreciable effect when coupled with the organic 
matter. In the soil alone, the increasing amounts of nitrates gave im- 
proved plant growth for the early part of the plant season, but these 
differences were obliterated when the crop matured, as was shown by the 
weights and nitrogen content of the crop. The differences in the four 
pot series just before their close are shown in plate 1. 



290 



WILLIAM ALBERT ALBRECHT 



Nitrates in soil growing soybeans. Before removing the plants at harvest, 
samples of soil were taken and nitrates determined by extraction and reduction 
with Devarda's alloy. Forty-gram samples of water-free soil were used. De- 
terminations on those pots with organic matter added were somewhat erratic 
and failed to check well in duplicate. Since large amounts of organic matter 
are known to interfere with reduction methods (9, 48) and give high results, 
full credence cannot be placed on the results from these soils with organic 
matter. Table 8 gives the results of the determinations of nitrates. 

TABLE 8 
Nitrate nitrogen in soil growing soybeans 



POT 


NITROGEN ADDED (POUNDS PER 2,000,000 POUNDS OF SOIL) 


MILLIGRAMS 

IN 40 GRAM 

SOIL 


MILLIGRAMS 
PER POT 


POUNDS PER 

2,000,000 

POUNDS or 
SOIL 


1 


None 


0.337 


28 


16.8 


2 


None 


0.366 


31 


18.3 


3 


None 


0.378 


32 


18.9 


4 


None 


0.337 


28 


16.8 


5 


10 pounds as nitrate 


0.397 


33 


19.8 


6 


10 pounds as nitrate 


0.378 


32 


18.9 


7 


50 pounds as nitrate 


0.337 


28 


16.8 


8 


50 pounds as nitrate 


0.397 


33 


19.8 


9 


150 pounds as nitrate 


1.353 


115 


67.6 


10 


150 pounds as nitrate 


1.533 


131 


76.6 


11 


1000 pounds as clover tops 


2.031 


176 


100.0 


12 


1000 ix)unds as clover tops 


0.816 


71 


40.0 


13 


2000 pounds as clover tops 


2.442 


216 


122.0 


14 


2000 pounds as clover tops 


5.835 


516 


291.0 


15 


3000 pounds as clover tops 


15.852 


1425 


792.0 


16 


3000 pounds as clover tops 


12.585 


1132 


629.0 


17 


4000 pounds as clover tops 


9.459 


864 


472.0 


18 


4000 pounds as clover tops 


9.757 


892 


487.0 


19 


5000 pounds as clover tops 


8.403 


780 


420.0 


20 


5000 pounds as clover tops 


8.722 


810 


436.0 



From the above table it is evident that the pots receiving the equivalent of 
10 and 50 pounds of nitrate nitrogen per acre contained no more nitrogen in 
this form at the close of the series than the pots left untreated. Evidently the 
plants consumed all that was applied, since soil conditions were unfavorable 
to denitrification, the other possible chance for nitrate removal. With the 
pots receiving the equivalent of 150 pounds per acre the conditions were dif- 
ferent and the nitrate content was much higher. The application of such a 
large amount, far above that needed for the plant growth, left some in the soil, 
since no leaching took place and the open soil prohibited denitrification. This 
would suggest that such a large apphcation suppHes more nitrogen than a 
single crop of soybeans can remove. 



SYMBIOTIC NITROGEN FIXATION 291 

In the soils with organic matter the figures indicate excessive amounts of 
nitrates, reaching the maximum in the pots receiving 3000 pounds of nitrogen 
as clover tops per 2,000,000 pounds of soil, and decreasing somewhat with the 
heavier applications. The high results may have been caused partly by the 
interference of the organic matter. 

Nodule production by soybeans. When the plants were harvested, the roots 
were removed from the soil as completely as possible and studied for nodule 
production. Pots 1 and 2, untreated, which had not been inoculated at the 
outset were still sterile in respect to the soybean bacteria, containing no nod- 
ules and showing no contamination from the inoculated pots all about them. 
This indicates that there is no great danger of inoculation by contamination 
in an experiment of short duration of this kind with soybeans when no special 
precautions are taken. In the pots in this series, treated with sodium nitrate, 
many good-sized nodules were present giving no visible effect from the nitrate 
treatment either on the size or on the number of the nodules, except perhaps in 
the two pots receiving the equivalent of 150 pounds of nitrogen per acre. In 
these the nodules were smaller, but this difference was offset by increased 
numbers. 

In the pot series treated with clover tops, nodule production decreased with 
added amounts of organic matter. However, in the soils with the highest ap- 
plication there was such a poor development of the plants that very few 
nodules could be expected. The low vitality and poor plant growth rather 
than any disturbing factors in the soil may have been the cause of insignificant 
nodule formation. There was no evidence to indicate that with healthy plants 
nodule production would have been prohibited by these large amounts of or- 
ganic matter. 

In the soil treated with such large amounts of clover tops as would give 5000 
pounds of nitrogen in 2,000,000 pounds of soil, the original composition and 
physical make-up were radically altered. Such modifications of soil condi- 
gions invited the infestation by a winged fungus gnat {Sciara mycetophilidae) 
which may have consumed and removed much of the nitrogen. The open soil 
structure and a plentiful food supply in the form of decaying organic matter 
furnished an excellent habitat for these small insects. The infestation origin- 
ated in the pots with the heaviest applications of organic matter, but spread 
to all pots so treated. Its duration was Hmited to a certain stage or period in 
the decomposition of the organic matter, and the insects were present only 
long enough to develop the larvae and pupae of one generation. They may 
have been a disturbing factor of which no account could be taken, and may 
have been partly responsible for the losses of nitrogen in these series. 

In tables 9, 10, 11 and 12 is given the nitrogen balance for the four pot series 
with soybeans. Many of the analytical data are omitted and only the sum- 
mation figures are given to show the balance between the total nitrogen in the 
soil and seed at the beginning, and that of the soil and crop at the close. Fig- 
ures given are the results of quadruplicate determinations in the soil, while in 

SOIL SCIENCE, VOL. IX, NO. 5 



292 



WILLIAM ALBERT ALBRECHT 



the crop the total material was analyzed. Four pots given in the first series 
are used as the checks for the remaining pot series. 

Following the tables is a graphical representation of the increases and losses 
in total nitrogen shown in the tables. The ordinates represent milligrams of 
nitrogen gained or lost while the abscissas represent nitrate nitrogen added to 
the soil in case of one curve, and the total nitrogen present in the soil at the 
outset for the remaining curves. 

Instead of showing an increase in nitrogen, most pots show a decrease. In- 
creases in nitrogen, when they occur, are insignificant as compared with the 

TABLE 9 

Nitrogen balance — Soybeans 

Pot series 1, soil treated with nitrates 



POT 


nitrate nitro- 
gen ADDED 


SAMPLE 
WATER- 
FREE SOIL 


NITROGEN 

IN 

SAMPLE 


NITRO- 
GEN IN 
POT AT 
CLOSE 


CROP 
WEIGHT 


NITRO- 
GEN IN 
CROP 


NITROGEN 

AT CLOSE 

(SOIL-f 

CROP) 


NITRO- 
GEN AT 
OUTSET 
(SOIL -t- 

seed) 


INCREASE 

OR 
FIXATION 


AVER- 
AGE 




mgm. 


gm. 


mgm. 


mgm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


1 


None (uninoc- 






















ulated) 


9.8394" 


2.8713^ 


9981= 


2.9 


36" 


10.34 


1117" 


-83 




2 


None (uninoc- 
ulated) 


9.8215 


2.9714 


1032 


4.0 


58 


1090 


1117 


-55 f 


-36 


3 


None 


9.8260 


2.9S48 


1040 


3.5 


98 


1138 


1117 


21 




4 


None 


9.8288 


2.8864 


1003 


3.85 


89 


1092 


1117 


-25 J 




5 


17.2 


9.7864 


2.8783 


1005 


3.85 


95 


1100 


1134 


-341 


-16 


6 


17.2 


9.8025 


2.9714 


1037 


4.75 


98 


11.35 


11.34 


-1/ 


7 
8 


86 
86 


9.8029 
9.8183 


2.9140 

2.8498 


1016 
992 


5.12 
4.25 


113 
98 


1129 
1090 


1203 
1203 


-74\ 
-113/ 


-93 


9 


258 


9.8087 


3.2243 


1124 


4.25 


124 


1248 


1375 


-1271 


-122 


10 


258 


9.8190 


3.1790 


1106 


3.70 


151 


1257 


1375 


-118/ 



* Average of four determinations, or duplicates made at two different times. 

^ Based on 3421 gm. water-free soil in each pot. 

° Found by analyzing entire crop, roots and tops. 

^ Based on the analyses of the soil at the outset giving 0.03136 per cent of nitrogen, or 
1072 mgm. of nitrogen in each pot with 3421 gm. of soil, and 5 seeds containing 45 mgm. of 
nitrogen. Additions of nitrate were equivalent to 17.2, 86 and 258 mgm. of nitrogen. 



losses. When the soil was treated with nitrates only, one of the duphcate pots 
showed an increase in nitrogen at the close over that present at the outset, 
but this increase was within the limit of difference between duplicate determi- 
nations which, when calculated per pot, was 60 mgm. of nitrogen. The limits 
of error in nitrogen per pot were less, however, than this figure, as a result of 
quadruphcate analysis. The largest loss of nitrogen was 127 mgm. As more 
nitrate nitrogen was added at the beginning, the loss at the close increased, 
corresponding closely to the amount added. This indicates that perhaps the 



SYMBIOTIC NITROGEN FIXATION 



293 



analytical procedure failed to detect the nitrate nitrogen added. Had the 
nitrate nitrogen been incorporated into the plant tissue as protein, it would 
certainly have been found; but it might be possible, in spite of all care in this 
respect, that such small amounts of nitrate were not detected by the analysis 
for total nitrogen at the close. Later crop series receiving far larger amounts 
of nitrogen as nitrate, failed to show any such discrepancies. 

TABLE 10 

Nitrogen balance — Soybeans 

Pot series 2, soil treated with clover tops 



POT 


NITRO- 
GEN 
ADDED 

AS 

CLOVER 

TOPS 


SAMPLE 

WATER - 

FREE 

SOIL 


NITROGEN 
IN SAMPLE 


SOIL IN 
POT 


NITRO- 
GEN IN 
POT AT 
CLOSE 


CROP 
WEIGHT 


NITRO- 
GEN IN 
CROP 


NITRO- 
GEN AT 

CLOSE 
(SOIL + 

CROP) 


NITRO- 
GEN AT 
OUTSET 

(soil -h 
seed) 


in- 
crease, 

OR 

fixation 


aver- 
age 




mgm. 


em. 


mgm. 


gm. 


mgm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


11 


1750" 


9. 8361 b 


7.7461b 


3480 


2740° 


2.85 


122 


2862 


2867 


-5\ 


79 


12 


1750 


9.8385 


8.0541 


3480 


2848 


4.70 


182 


3030 


2867 


163/ 


13 


3500 


9.8279 


12.3984 


3539 


4464 


4.15 


189 


4653 


4617 


36\ 


90 


14 


3500 


9.8216 


12.7212 


3539 


4583 


4.05 


179 


4762 


4617 


145 J 


15 
16 


5250 
5250 


9.7951 
9.7905 


15.4721 
16.3141 


3598 
3598 


5683 
5995 


3.55 
2.70 


199 
154 


5882 
6149 


6367 
6367 


-485 \ 
-218J 


-351 


17 

18 


7000 
7000 


9.7815 
9.8248 f 


19.9884 
19.3026 


3657 
3657 


7473 
7184 


1.85 
1.70 


131 
99 


7604 

7283 


8117 
8117 


-513\ 
-834j 


-673 


19 
20 


8750 
8750 


9.7879 
9.8146 


23.2323 
23.3436 


3716 
3716 


8820 
8838 


1.25 
1.20 


91 

82 


8911 
8920 


9867 
9867 


-956\ 
-947 J 


-951 



» Clover tops added were figured on the basis of 3500 gm. of soil per pot instead of 3421, 
hence are not truly equivalent to the number of pounds per 2,000,000 pounds of soil as the 
increments were intended. 

^ Average of four determinations, or duplicates made at two different times. 

" Based on water-free soil in pot as given in fifth column. 

"^ Found by analyzing entire crop, roots and tops. 

» Based on the analyses of the soil at the outset giving 0.03136 per cent of nitrogen, or 
1072 mgm. of nitrogen in each pot with 3421 gm. of soil, and 5 seeds containing 45 mgm. of 
nitrogen. 

' Two determinations only. 

In the three series receiving organic matter alone and organic matter in 
conjunction with 10 and 50 pounds of nitrogen as nitrate, there was an increase 
in nitrogen for all pots receiving clover tops equal to 1000 pounds of nitrogen 
per acre, and for two pots treated with organic matter equivalent to 2000 
pounds of nitrogen. All other pots in these three series showed decided losses. 
In the pots with the equivalent of 5000 pounds of nitrogen added as clover 
tops this loss ran as high as 1150 mgm. of nitrogen in some few cases. 



:.:^K 



294 



WILLIAM ALBERT ALBRECHT 



These large losses must have resulted from the very rapid decomposition of 
the fresh organic matter and doubtless the nitrogen of the clover tops escaped 
as gaseous ammonia. The excessive application encouraged rapid decomposi- 
tion, and with 320 gm. of clover tops incorporated in 3421 gm. of soil, the pro- 
portion of soil may have been too small to absorb all the ammonia. The losses 
increased fairly regularly with larger applications of clover tops, though in no 
definite mathematical relation. 

TABLE 11 
Nitrogen balance — Soybeans 
Pot series 3, soil treated with clover tops and 10 pounds of nitrate nitrogen 



21 

22 
23 
24 
25 
26 
27 
28 
29 
30 



NITROGEN ADDED 



mgm. 
fl750^ as clover 
< tops and 
[17.2 as nitrate 

3500 as clover 

tops and 
17.2 as nitrate 

(5250 as clover 
tops and 
17.2 as nitrate 

[7000 as clover 
\ tops and 
17.2 as nitrate 

8750 as clover 

tops and 
17 . 2 as nitrate 









w 


H 


5 

14 


nd 


H O 


(4 


SAMPLE 
WATER- 


NITROGEN 


H 
O 




H 
O 


< o 

Z— bT 

W M O 


Www 


w2 


FREE 


IN SAMPLE 


Z 




^ 


O P< 


o a Bi 


Sjs w 


<% 


SOIL 






2 f-' 

ns o 


B. 


O O 
ex! a! 


o o u 

OS Hi 1 


Bi P , 
H + 


gS 












H U 


oE 






LO 


Z 


O 


Z 


Z 


z 




gm. 


mgm. 


gm. 


mgm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


9.7732b 


7.9007b 


3480 


2813 « 


3.85 


144 d 


2957 


2884" 


73] 


9.7804 


8.1348 


3480 


2894 


4.1 


167 


3061 


2884 


177J 


9.7684 


11.8428 


3539 


4290 


4.2 


167 


4457 


4634 


-177] 


9.7537 


12.1518 


3539 


4409 


3.9 


169 


4578 


4634 


-56] 


9.7672 


14.8770 


3598 


5480 


2.2 


123 


5603 


6384 


-781] 


9.7629 


15.7567 


3598 


5806 


2.0 


126 


5932 


6384 


-452 J 


9.7648 


18.6387 


3657 


6980 


1.9 


105 


7085 


8134 


-949] 


9.7642 


19.2363 


3657 


7204 


1.85 


126 


7330 


8134 


-8O4J 


9.7626 


22.6532 


3716 


8628 


0.05 


73 


8701 


9884 


-1183] 


9.7556 


22.4843 


3716 


8653 


1.80 


110 


8763 


9884 


-II21J 



125 



-116 



-616 



-876 



-1152 



* Clover tops and nitrate added were figured ou the basis of 3500 gm. of soil per pot in- 
stead of 3421, hence are not truly equivalent to the number of pounds per 2,000,000 pounds 
of soil as the increments were intended. 

b Average of four determinations, or duplicates made at two different times. 
" Based on weights of soil given in fifth cohimn. 
^ Found by analyzing entire crop, roots and tops. 

* Nitrogen in soil 1072 mgm., nitrogen in seed 45 mgm., nitrogen m 64.59 gm. clover tops 
equivalent to 1000 pounds per acre 1750 mgm., and nitrogen as nitrate 17.2 mgm. 



From the data of this series, one cannot say with any great certainty that 
the soybeans so grown used atmospheric nitrogen. The treatment of nitrate 
apparently prevented an increase in nitrogen while the treatment of clover tops 
equal to 1000 pounds of nitrogen gave an increase of nitrogen. This must 



SYMBIOTIC NITROGEN FIXATION 



295 



have come from the air in the form of nitrogen fixation. The fact that there 
is a loss of total nitrogen in the system does not deny the possibiHty of the 
plants fixing nitrogen, for the legume may have used atmospheric nitrogen at 
the same time this escape from the soil was taking place. 

The crop growth on all these series was small, and the total nitrogen in the 
crop of any pot never exceeded 200 mgm. With such small plant growth no 



TABLE 12 

Nitrogen balance — Soybeans 
Pot series 4, soil treated w ith clover tops and 50 pounds of nitrate nitrogen 



POT 


NITROGEN ADDED 


SAMPLE 
WATER- 
FREE 
SOU 


NITROGEN 
IN S.\MPLE 


H 

O 

g 

iJ 
)-i 
O 
tn 


a < 

!5 


H 

m 
a 

p< 
o 

O 


!5 

OS as 
H o 


< O 

z-^7: 

W H O 

o S « 

O o " 
H + 


1^ 

H 3 

« » i_ 


°Z 
Z 


1 

< 




mgm. 


gw. 


mgm. 


gm. 


mgm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


31 


[1750* as clover 
\ tops and 


9.7603b 


8.4575b 


3480 


3015° 


3.6 


154d 


3169 


2953" 


216 


114 


32 


86 as nitrate 


9.7656 


7.9314 


3480 


2826 


3.3 


140 


2966 


2953 


I3J 




33 


3500 as clover 
tops and 


9.7694 


12.0295 


3539 


4357 


3.25 


144 


4501 


4703 


-202] 


-187 


34 


86 as nitrate 


9.7561 


12.0698 


3539 


4378 


3.05 


152 


4530 


4703 


-I73J 




35 


5250 as clover 
tops and 


9.7481 


15.0377 


3598 


5550 


1.5 


93 


5643 


6463 


-820] 


-490 


36 


86 as nitrate 


9.7474 


16.7461 


3598 


6181 


1.9 


122 


6303 


6463 


-I60J 




37 


[7000 as clover 
tops and 


9.7386 


19.6224 


3657 


7368 


1.5 


101 


7469 


8203 


-734] 


-950 


38 


86 as nitrate 


9.7546 


18.4956 


3657 


6934 


1.65 


103 


7037 


8203 


-1166J 




39 


('8750 as clover 


9.7601 


23.3127 


3716 


8875 


0.9 


47 


8922 


9953 


-10311 






tops and 


















I 


-1148 


40 


86 as nitrate 


9.7570 


22.6049 


3716 


8609 


1.05 


79 


8688 


9953 


-1265 J 





* Clover tops and nitrate added were figured on the basis of 3500 gm. of soil per pot in- 
stead of 3421, hence are not truly equivalent to the number of pounds per 2,000,000 pounds 
of soil as the increments were intended. 

b Average of four determinations or duplicates made at two different times. 
" Based on weights of soil given in fifth column. 
^ Found by analyzing entire crop, roots and tops. 

• Nitrogen in soil 1072 mgm., nitrogen in seed 45 mgm. , nitrogen in 64.59 gm. clover tops 
equivalent to 1000 pounds per acre 1750 mgm., and nitrogen as nitrate 86 mgm. 

great fixation was possible for the fixation of atmospheric nitrogen consists in 
the utiHzation of this form of nitrogen by the plant for tissue building, and 
unless plant growth is significant, no marked use of atmospheric nitrogen can 
be expected. The conditions of this part of the experiment gave too narrow 
a margin between the possible nitrogen fixed and the limits of variation, and 



296 



WILLIAM ALBERT ALBRECHT 



even though increases were shown in some cases, they failed to give assurance 
of any significant fixation. Unless a reasonable amount of fixation was taking 
place, the influence of the amount of nitrogen in the soil upon it could not be 
measured. 



leo 




Fig. 1. Graphs Showing Nitrogen Fixation by Soybeans on Soils Treated 
WITH Nitrates, Clover Tops, and with Nitrates and Clover Tops 

Series 2 (Cowpeas) 

For the second crop series cowpeas were grown in the hope that they would 
be less subject to the infestation by insects, more apt to do well under green- 
house conditions and more able as nitrogen fixers. Only two soil treatments 
were employed, one in which increasing amounts of nitrate nitrogen were 



SYMBIOTIC NITROGEN FIXATION 297 

added and the other one in which the nitrogen had been increased by organic 
matter. 

Some of the original soil left over when the previous crop series was made up 
and stored in the dry condition was used for the treatment with nitrate ni- 
trogen. The equivalent of 3300 gm. of water-free soil was weighed into each 
pot and enough sodium nitrate added in a solution to give increases in nitrogen 
equivalent to 10, 25, 50, and 100 pounds of nitrogen in 2,000,000 pounds of 
soil. 

For the series whose nitrogen increase was in the form of organic matter, 
the soils used in the previous series in the pots 1 to 4 and 11 to 20, inclusive, 
were used again. Pots 1 to 4 had received no treatment with nitrogen and 
again served as checks. Pots 11 to 20 were also used without modification, 
save that the amount of soil in each pot was limited to 3300 gm. on the water- 
free basis. The nitrogen content in these soils no longer showed increments of 
1000 pounds per 2,000,000 pounds of soil as a result of losses while growing 
soybeans, but were used because their differences in nitrogen content were very 
marked and they were similar in all respects except this one. 

Plant-foods other than nitrogen were supplied in a soluble form. Calcium 
carbonate was added at the rate of 2 tons per 2,000,000 pounds. 

Six cowpeas of uniform weight and known nitrogen content were planted 
in each pot and later when the plants were well started were reduced to five. 
The nitrogen added by the five cowpea seeds was equivalent to 43 mgm. 
During the early spring the pots were kept in the greenhouse but later were 
kept outdoors in a screened area. No difficulty in germination was experi- 
enced, and the growth was decidedly better than that of the soybeans. 

Differences in these two series were soon noticeable. For the series receiving 
nitrate the plants were taller, the leaves larger and the color deeper where 
greater amounts of nitrate were added. These differences later disappeared 
so that by the time of harvest, there were no significant variations within the 
entire series, either in crop weight or total nitrogen. 

The soil series treated with nothing but organic matter and which had been 
previously used for soybeans, gave a decidedly better growth of cowpeas than 
the series treated with nitrates. The differences are shown in plates 2 and 3. 
In this series the early growth was best in the pot receiving 1000 pounds of 
nitrogen per acre as clover tops. There was a decrease in growth with the 
increase of added organic matter, but even all these were equal to the check, 
receiving no organic matter. In the latter part of the growing season these 
differences were reversed, and the largest crop yields and the largest total 
amounts of nitrogen in the crop were produced on the pots receiving most 
organic matter. The crops were grown for about 135 days and then harvested. 
Some plants had produced blossoms and a few had set pods. The fact that 
the pots were much shaded by some large trees lengthened the vegetative 
period and delayed seed development. 



298 



WILLIAM ALBERT ALBRECHT 



When the crops were harvested the roots were removed as completely as 
possible and carefully examined for nodules. It is important to note that 
the nitrate series produced many nodules, even with 100 pounds of nitrogen in 
this form. Where organic matter was added, the nodules were most numerous 
in the lesser appHcations but were larger in size as the applications increased. 
There were no indications in any pots that insufficient nodules were present 
for nitrogen fixation. If the presence of the nodule is certain evidence that 
atmospheric nitrogen is used by the plant, then both these soil series permitted 
fixation to take place in all pots regardless of treatments. 

Determinations of nitrate nitrogen in the soils in all the pots were made 
just before the plants were harvested. In the soil treated with sodimn nitrate, 
no nitrogen in this form was found. The determinations of nitrates gave re- 

TABLE 13 

Nitrogen balance — Cowpeas I 

Pot series 1, soil treated with nitrates 





o 

H O 
Z 




5 
S 


o < 
O H 
Bi O 
H 0< 


WEIGHT OF 
CROP 


NITROGEN IN 
THE CROP 


5S 
Z-S-r- 


<^_ 

Z H O 

H M H 
O m W 
O H "2 
« Pi 
H OT 

Z 


°Z 
U h 

S 


m 


H 
O 




o 


O 

H 


o 
o 
OS 


< 




mgm. 


gm. 


mgm. 


mgm. 


gm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


la 


None 


9. 7673 "^ 


3.27S5« 


1106^ 


15.75 


8.855 


415 


152 


1673 


1077° 


596\ 


688 


2a 


None 


9.6763 


3.7410 


1275 


14.75 


9.435 


418 


164 


1857 


1077 


780/ 


3a 


16.5 


9.7201 


3.3414 


1134 


14.80 


7.820 


306 


139 


1579 


1093 


486\ 


533 


4a 


16.5 


9.7034 


3.1594 


1074 


16.40 


7.800 


440 


159 


1673 


1093 


580/ 


5a 


41.0 


9.6933 


3.1163 


1061 


14.45 


7.145 


292 


147 


1500 


1118 


382 \ 


512 


6a 


41.0 


9.7568 


3.3418 


1130 


17.65 


7.781 


445 


185 


1760 


1118 


642/ 


7a 


82.0 


9.7691 


2.9373 


992 


16.15 


8.408 


404 


162 


1558 


1159 


399 \ 


453 


8a 


82.0 


9.7215 


3.1031 


1053 


16 65 


7.675 


445 


168 


1666 


1159 


507/ 


9a 


157.0 


9.7441 


3.0102 


1019 


15.45 


6.798 


347 


124 


1490 


1234 


256\ 


264 


10a 


157.0 


9.7641 


2.9108 


983 


14.80 


5.610 


385 


138 


1506 


1234 


272/ 



* Average of three determuiations. 
^ Calculated on the basis of 3300 gm. of water-free soil. 

" Based on 3300 gm. of water-free soil with 0.03136 per cent of nitrogen, or 1077 mgm. of 
nitrogen, and five cowpea seeds with 43 mgm. of nitrogen. 

suits no larger than those of the blank determinations. For the series with the 
organic matter, this treatment interfered with the analytical procedure for 
nitrates so that no definite statement of the amounts of nitrate present can 
be made. Results were erratic, but indications pointed to the presence of 
comparatively large amounts of nitrate nitrogen. Evidently nitrification 
was going on and nitrates were present in the soil but this had not prohibited 
the production of many large-sized nodules on the roots of the cowpeas. 

The harvested crops were thoroughly dried, weighed and later ground to 
permit uniform sampling. The crop growth was too large to be analyzed in 
total and only triphcate 2-gm. samples were used. The soil was air-dried, 



SYMBIOTIC NITROGEN FIXATION 



299 



ground and sampled. Analyses were made by the same methods used in the 
previous series, but instead of using four samples, only triplicate determina- 
tions were made. On the basis of these analyses, the total nitrogen in the soil 
and the crop at the close of the series was calculated. 

Tables 13 and 14 give the nitrogen balance for the second crop series with 
cowpeas. Only the summations of analytical data are given. Since the soils 
in pots 1 to 4 and 11 to 20 were analyzed at the close of the soybean series no 
analyses were made on these soils at the beginning of this series, but the amount 

TABLE 14 

Nitrogen balance — Cowpeas I 

Pot series 2, soil treated with clover tops 





z 
w a 

o o 


H 


g 

■is 
s 


O H 

1' 


WEIGHT OF CROP 


NITROGEN IN 
THE CROP 


5^ 

W M O 
O S M 
O o tJ 


Www 

g§ + 

s 


w-2 
5 


1 


a. 
o 

H 


o 
o 

PS 


a. 


o 
o 

Pi 




Ihs. 


gm. 


mgm. 


mgm. 


gm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


1 


None 


9.7140" 


3.5308'' 


1199>' 


21.70 


6.976 


604° 


135 d 


1938 


1006" 


9321 


2 


None 


9.7324 


3.4744 


1178 


19.65 


6.040 


559 


126 


1863 


1041 


822 j 


3 


None 


9.7423 


3.3086 


1120 


18.75 


7.170 


504 


157 


1779 


1045 


734\ 


4 


None 


9.7026 


3.2257 


1097 


21.90 


10.182 


502 


178 


1777 


1012 


765/ 


11 


995' 


9.7421 


6.8328 


2314 


34.75 


11.705 


1066 


201 


3581 


2641 


941 \ 


12 


995 


9.7419 


7.0649 


2393 


35.30 


9.525 


1032 


205 


3630 


2744 


886J 


13 


1960 


9.7324 


9.5746 


3246 


33.00 


10.550 


977 


192 


4415 


4206 


2091 


14 


1960 


9.7412 


9.5912 


3249 


39.75 


12.711 


1125 


209 


4576 


4317 


259/ 


15 


2890 


9.7462 


11.5307 


3904 


39.20 


11.959 


un 


204 


5325 


5255 


70l 


16 


2890 


9.7514 


11.6965 


3958 


36.45 


12.270 


1124 


226 


5308 


5541 


-223] 


17 


3790 


9.7549 


13.6027 


4601 


43.65 


9.975 


1256 


153 


6010 


6786 


-776\ 


18 


3790 


9.7468 


13.4631 


4558 


37.25 


8.347 


1290 


189 


6037 


6526 


-489/ 


19 


4660 


9.7461 


15.6151 


5287 


45.70 


10.255 


1594 


125 


7006 


7875 


-8961 


20 


4660 


9.7516 


15.9400 


5394 


41.45 


8.215 


1386 


153 


6933 


7891 


-958J 



mgm. 

877 
749 

913 

234 

-81 

-632 

-927 



" Average of three determinations. 

^ Calculated on the basis of 3300 gm. of water-free soil. 

" Found by analyzing three 2-gm. samples and calculating from the weight of tops. 

^ Found by analyzing the entire root system. 

® Based on analysis made at the close of the soybean series (data in table 10). 

' The additions of nitrogen as clover tops made on these soils previous to the growth of 
soybeans are given as pounds of nitrogen added per 2,000,000 pounds of the mixture of soil 
and clover tops. 



of nitrogen in 3300 gm. of soil was calculated from the preceding analyses. 
The soil which had been stored and used in this series was analyzed and found 
to contain the same amount of nitrogen as at the beginning of the soybean 
series, or 0.03136 per cent of nitrogen on the water-free basis, which gave 1034 
mgm. of nitrogen per pot. The crop weights and the amounts of nitrogen in 
the crop are given separately as tops and roots. 



300 



WILLIAM ALBERT ALBRECHT 



Following the tables is a graphical representation of the nitrogen balance in 
the series (fig. 2). 

In the series treated with sodium nitrate the amounts of total nitrogen 
present at the close were larger in every pot than those present at the outset. 



1000 



800 



600 



400 



N 200 




200 



400 



600 



800 



TT 



10(10 



2r 



aoDo 



60 



51 )90 



Po 
soi 



IL 



40 30 



ids ni 
trea 



trog 
ted 1 

50,30 



en per 
with c 



^acre li 
Lover tcps. 



IDO 
\ Potmds nitrogen adjded per 
X acre as nitrate 



Fig. 2. Graphs Showing Nitrogen Fixation by Cowpeas on Soils Treated 
WITH Nitrates, and with Clover Tops 

This indicates decidedly that nitrogen was fixed from the atmosphere. The 
amounts so obtained were very significant, and in every case far above the 
limits of variation in analytical determinations. The average nitrogen in- 



-.n>^ 



SYMBIOTIC NITROGEN FIXATION 301 

crease, or nitrogen fixation, from duplicate pots shows a gradual decrease with 
increase in nitrate nitrogen added to the soil. The largest fixation appeared 
in the pots whose soil received no nitrogen additions and was reduced about 
the same in the two soils treated at the rate of 10 and 20 pounds of nitrate 
nitrogen per 2,000,000 pounds of soil. On the soil treated with the equivalent 
of 100 pounds of nitrate nitrogen the fixation was about 38 per cent of that 
where no treatment was applied. The general decrease in fixation with in- 
creased treatment would lead one to believe that one or two more increments 
beyond the appHcation of 100 pounds would have prohibited fixation com- 
pletely. The table indicates that increasing amounts of nitrate nitrogen in 
the soil reduce nitrogen fixation by cowpeas, and may perhaps prohibit it, but 
the amounts required for significant reduction of this process are far greater 
than ever occur in a soil or are ever applied. 

The series treated with organic matter and previously used for growing soy- 
beans had some pots which failed to show any increase in total nitrogen at 
the close over that at the outset. Significant increases were obtained for the 
check pots and those originally treated with the equivalent of 1000 and 2000 
pounds of nitrogen per acre, but for the rest of the treatments a negative 
fixation, or loss, took place. This was greater with larger applications of ni- 
trogen. It is highly probable that the organic matter put on the soil in such 
heavy applications one season previously was still undergoing decomposition 
rapidly enough to lose nitrogen as ammonia or as gaseous nitrogen. Decom- 
position had not yet gone far enough to change all the organic matter of such 
heavy applications into a more stable form, from which no losses could take 
place. Such losses from the total nitrogen present in soil and crop at the close, 
as compared with that present in soil and seed at the beginning, do not prove 
that the plant failed to draw on the atmosphere for some of its nitrogen supply. 
Nodules were plentiful in the soils receiving the heaviest applications of organic 
matter and some use may well have been made of gaseous nitrogen. Any 
fixation that could have taken place was offset by the losses from the soil, 
and could not be detected in this method of determination. 

Considering the averages for duplicate pots, the amounts fixed in the check 
pots 1 and 2, which had previously grown the uninoculated soybeans, were 
larger than that fixed by pots 3 and 4, on which the soybeans were inocu- 
lated. This difiference between the two sets of check pots resulted from a 
larger amount of nitrogen being present in both the crop and the soil in the 
first two pots. The reverse was true, however, of the total crop weights, 
largely because of greater root development in pots 3 and 4. In pots 11 and 12 
the average fixation was the largest in the series. This would indicate that 
the treatment with organic nitrogen corresponding to 1000 pounds per acre 
was beneficial to nitrogen fixation by cowpeas in a soil so low in this element as 
this soil was. This statement cannot be made, however, for all the higher 
applications. A decided increase was shown by the pots receiving 2000 pounds 
of nitrogen per acre, but those receiving more showed a loss rather than a gain. 



302 WILLIAM ALBERT ALBRECHT 

Evidently the lower applications do not prohibit nitrogen fixation, but whether 
the larger ones prohibit the process cannot be said from the preceding data. 
The losses coming doubtlessly from the decaying organic matter in the soil 
more than balance any nitrogen from the air added to the plant. In terms of 
total nitrogen in the soil rather than nitrogen added, those pots showed nitro- 
gen fixation whose soils contained the equivalent of approximately 625, 1625 
and 2600 pounds of nitrogen per 2,000,000 pounds. From this it may be 
safely said that a soil with these amounts of nitrogen does not prohibit the 
symbiotic nitrogen fixation even when 2000 pounds of nitrogen were in a 
readily decomposable form. The soils with higher nitrogen content failed to 
show similar results because the large amount of nitrogen applied to the soil 
was partly lost in a volatile form. 

Series 3 (Cowpeas) 

The third crop series was very similar to the second, with a few modifica- 
tions. It included one series of pots with soils treated with nitrates and an- 
other series with soils treated some time previously with organic matter in the 
form of clover tops. 

For treatment with nitrates some soil saved by careful storage from the 
first crop series of soybeans was used. Sodium nitrate was added in solution 
to increase the nitrogen content at increments equivalent to 50, 100, 150, 200 
and 250 pounds of nitrogen per 2,000,000 pounds of soil. Two additional 
pots receiving the equivalent of 50 pounds of nitrate nitrogen were included, 
but this was added in three applications at intervals extending over the greater 
part of the growing period. Three kilograms of soil were used per pot. 

For soils whose nitrogen was increased by organic matter, those pots used 
in both preceding crop series were used again without change, save that the 
amounts of soil were less, and only 2800 gm. of water-free soil were weighed into 
each pot. The nitrogen content of these soils did not increase in constant 
amounts for the series, but corresponded very closely to amounts equivalent 
to 625, 1625, 2600, 3250, 4025 and 4775 pounds of nitrogen per 2,000,000 
pounds of soil. These soils had been made up originally with enough clover 
tops to give a series whose nitrogen content increased by units of 1000 pounds, 
but the losses during the two preceding crop series reduced it to the above 
figures. It was thought advisable to use these soils again since their nitrogen 
content was accurately known, and the growth of two crops allowed sufficient 
time to permit decomposition of the organic matter to have gone far enough 
to prohibit further losses in volatile nitrogen. 

The soils were treated with calcium carbonate at the rate of 2 tons per 2,000- 
000 pounds of soil. Phosphorus, potassium and other mineral plant-food 
elements were applied in solution when the dry soil was first moistened and 
then at intervals during the crop growth. All soils were treated similarly in 
this respect. Attempts were made to keep the moisture content of the soil 



SYMBIOTIC NITROGEN FIXATION 303 

at optimum by weighing the pots, but this method was inaccurate when the 
plants became larger, so that water was applied at intervals to keep the mois- 
ture content near what seemed optimum. 

Cowpeas were grown as the crop. Six seeds weighing in total an average of 
1.109 gm. and containing 38 mgm. of nitrogen were planted in each pot. As 
the seedhngs were well started, one was removed to leave five vigorous plants. 

The growth of the crop was good. No difi&culty was experienced in germi- 
nation, and though the early growth was somewhat slow, the remainder of 
the growing season found the plants doing well. Differences due to treatment 
were manifested early. With increased amounts of nitrate applied the plants 
were taller, of a deeper color and the leaves more fleshy, but these marked 
differences disappeared later. The plants on soils treated with organic matter 
were larger and of deeper color as the nitrogen content of the soil increased. 
These variations became less prominent and were completely lost before the 
close of the experiment. The total plant growth was similar to that in the 
preceding crop series, but with less variations. Plate 4 illustrates the crop 
growth of the two series just before harvest, which was about 22 weeks after 
planting. 

Shortly before the crops were harvested, several blossoms were formed but 
were removed to keep these few plants from setting seed. All blossoms and 
fallen leaves were collected and saved for analysis. The harvested plants 
were dried in the greenhouse, and later weighed and thoroughly ground for 
analysis. 

The roots were carefully removed, examined for nodules and thoroughly 
dried. No great differences in nodule production were evident as correlated 
with the treatment. There were no significant differences shown by the nod- 
ules in the soils treated with nitrates. Nodules were numerous in all pots and 
especially so in those receiving the equivalent of 250 pounds of nitrogen per 
2,000,000 pounds of soil. There was no evidence in either of the duphcate 
pots that the appHcation of such large amounts of nitrate interfered with 
nodule production. This application was equivalent to 12.5 mgm. of nitrogen 
per 100 gm. of soil and its failure to suppress nodule growth agrees well with 
the results of Fred and Graul (17) who found that 10 mgm. of nitrate nitrogen 
per 100 gm. of Miami silt loam were required before nodules of alfalfa and 
crimson clover were decreased by such treatment. Evidently the application 
in the yellow silt loam used in this case was still less than was required for 
serious effect on nodule production. 

Nodules were present in all pots treated with organic matter and gave indi- 
cations of being larger and better distributed within those soils receiving the 
heaviest apphcations This may have been due to the better physical condi- 
tion and greater aeration caused by such treatment. These results are not in 
accord with those found by Frank (14) for red clover, on which the nodules 
were fewer and smaller in the soil rich in organic matter. 



304 



WILLIAM ALBERT ALBRECHT 



At the close of this series, determinations of nitrate nitrogen were made on 
all the soils. Those treated with nitrate failed to show any nitrogen of this 
form present in the soil. This result is quite different from that found for 
soybeans, in which case an application of 258 mgm. per pot left much nitrate 
in the soil. The total nitrogen in the crop of soybeans was only 150 mgm. 
With the profuse growth of cowpeas in this series the application of 390 mgm. 
of nitrogen as nitrate was far below the nitrogen in the crop and might well be 
expected to be removed on account of its solubility. The removal was so 
complete that no more than a trace of nitrate was found in any of the soils. 



TABLE 15 

Nitrogen balance — Cowpeas II 

Pot series 1, soil treated with nitrates 





A 


g 


^ 


H 
55 O 


WEIGHT OF 


NITROGEN IN 


H^ 


H O 


B) 






< V. 

HO 


<t3 


u a< 


2 H 

a o 


CROP 


THE CROP 


< o 

w w o 
o S <x 
o o u 




wS 


H 


g 


o 


1 


a 
o 


1 


a 


Pt 


a 


<. 


S5 


z 


H 


« 


H 


oi 


z 


z 


z 


< 




mgm. 


gm. 


mgm. 


mgm. 


gm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


lb 


None 


9.7363" 


3.7909'* 


11681- 


32.30 


10.230 


778° 


153 


2099 


965 d 


1134\ 


1212 


2b 


None 


9.7455 


3.8321 


1179 


36.35 


11.740 


909 


168 


2256 


965 


1291 J 


3b 


75 


9.7396 


3.8543 


1187 


34.53 


8.527 


884 


146 


2217 


1040 


1177\ 


1125 


4b 


75 


9.7428 


3.7909 


1167 


31.55 


9.196 


800 


147 


2114 


1040 


1074/ 


5b 


150 


9.7419 


3.7223 


1146 


33.88 


10.374 


828 


177 


2151 


1115 


10361 


1248 


6b 


150 


9.7414 


4.1902 


1290 


41.23 


14.539 


1109 


177 « 


2576 


1115 


1461/ 


7b 


225 


9.7477 


3.7863 


1165 


36.38 


12.709 


904 


196 


2265 


1190 


10751 


1185 


8b 


225 


9.7547 


4.0892 


1257 


38.03 


9.569 


1064 


165 


2486 


1190 


1296/ 


9b 


315 


9.7486 


3,9412 


1212 


37.08 


10.756 


968 


175 


2355 


1280 


10751 


1092 


10b 


315 


9.7535 


4.1229 


1268 


36.78 


12.215 


929 


192 


2389 


1280 


1109/ 


lib 


390 


9.7453 


3.9108 


1203 


38.53 


9.831 


1130 


159 


2492 


1355 


1137\ 


1187 


12b 


390 


9.7444 


4.0892 


1258 


37.28 


9.509 


1138 


197 


2593 


1355 


1238J 


13b 


75' 


9.7354 


3.8368 


1182 


22.83 


11.868 


559 


179 


1920 


1040 


8801 


935 


14b 


75 


9.7447 


3.9850 


1226 


27.41 


10.411 


651 


153 


2030 


1040 


990/ 



* Average of three determinations. 
^ Calculated on the basis of 3000 gm. of water-free soil. 

■= Found by analyzing three 2-gm. samples and calculating from the weight of tops. 
^ Calculated on the basis of 3000 gm. of water-free soil with 0.0311 per cent of nitrogen, 
or 933 mgm. of nitrogen, and five cowpea seeds with 32 mgm. of nitrogen. 
® Part of roots lost, and duplicate pot was used for data. 
' Applied as three applications at intervals during the growth of the crop. 

For the organic matter series about the same conditions obtained. No sig- 
nificant amounts of nitrates were present in any pots and only traces were 
found in those which had originally received the equivalent of 5000 pounds of 
nitrogen per acre as clover tops. Evidently the organic matter had undergone 
sufficient changes so that it no longer contained volatile organic compounds 
which interfered with the reduction method employed for determining ni- 
trates. Nitrification may have been going on but not at sufficient rates to 



SYMBIOTIC NITROGEN FIXATION 



305 



permit measurable accumulations above the consiunption by the plants. 
The rapid growth of the plants might well have removed all the nitrates formed 
by rapid nitrification processes. Since the determinations failed to show 
nitrates present, no tabulations of data are submitted. 

To determine the total nitrogen in the crop and soil at the close, the proced- 
ure of the previous series was followed. All possible precaution was exercised 
to eliminate errors and maintain uniform results throughout. Tables 15 and 
16 give the data of this series, showing the increase in total nitrogen at the 
close over that present at the beginning. 

TABLE 16 

Nitrogen balance — Cowpeas II 

Pot series 2, soil treated with clover tops 





55 

H O 
O W 
O « 

a a 

H < 
S 


W 


5 


O H 

S 


WEIGHT OF 
CROP 


NITROGEN IN 
THE CROP 


W M O 
O u B« 
O o o 


si 

U Ul H 
OHM 

H + 


« a 


O 


o 
H 


o 
o 


e5 


o 
o 




Ihs. 


gm. 


mgm. 


mgm. 


gm. 


gm. 


mgm. 


mgm. 


mgm. 


mgm. 


mgm. 


1 


None 


9.7280" 


4.1271" 


1187 b 


34.58 


6.886 


901" 


136d 


2224 


1049 » 


1175\ 


2 


None 


9.7293 


4.0034 


1152 


32.40 


10.846 


810 


169 


2131 


1030 


1101/ 


3 


None 


9.7392 


3.7909 


1089 


27.20 


9.466 


619 


162 


1870 


982 


888\ 


4 


None 


9.7321 


3.7020 


1064 


25.00 


8.044 


607 


148 


1819 


961 


858/ 


11 


995' 


9.7427 


7.2487 


2083 


35.00 


6.378 


1037 


137 


3257 


1995 


1262\ 


12 


995 


9.7498 


7.4031 


2126 


39.55 


5.951 


1127 


131 


3384 


2062 


1322 J 


13 


1960 


9.7454 


9.7580 


2803 


36.55 


7.379 


1160 


145 


4108 


2786 


1322 \ 


14 


1960 


9.7398 


9.7739 


2809 


39.40 


6.727 


1125 


122 


4056 


2788 


1268 J 


15 


2890 


9.7481 


11.9310 


3427 


36.93 


6.481 


1039 


124 


4590 


3344 


1246\ 


16 


2890 


9.7576 


11.8676 


3405 


33.93 


6.014 


1152 


109 


4666 


3390 


1276/ 


17 


3790 


9.7543 


14.1009 


4047 


35.75 


6.666 


1012 


117 


5176 


3946 


1230\ 


18 


3790 


9.7608 


13.3840 


3839 


38.26 


8.482 


1130 


131 


5100 


3900 


1200/ 


19 


4660 


9.7597 


15.8616 


4550 


32.61 


9.492 


927 


132 


5629 


4517 


11121 


20 


4660 


9.7553 


15.7601 


4523 


39.28 


8.595 


1136 


148 


5807 


4609 


1198/ 



mgm. 
1138 

873 

1292 
1295 
1261 
1215 
1155 



* Average of three determinations. 

'' Calculated on the basis of 2800 gm. of water-free soil. 

" Found by analyzing three 2-gm. samples and calculated from weight of tops. 

^ Found by analyzing entire root system. 

^ Based on analyses made at the close of the preceding cowpea series. Data in table 14 
figured for 2800 gm. of water-free soil and five cowpea seeds with 32 mgm. of nitrogen. 

' The additions of nitrogen as clover tops made on these soils previous to the growth of 
soybeans are given as pounds of nitrogen added per 2,000,000 pounds of the mixture of soil 
and clover tops. 

The above data show most decidedly that the addition of nitrates in even 
larger amounts than were used in the previous series does not prohibit an in- 
crease of nitrogen fixation. The use of this form of nitrogen in the previous 
test with cowpeas gave a gradual decrease of fixation as the application of 
nitrate increased. In that series nitrate equivalent to 100 pounds per 2,000,000 



306 WILLIAM ALBERT ALBRECHT 

pounds of soil permitted only 38 per cent as much fixation as no treatment, 
hence it suggested that double the appHcation would prohibit fijcation com- 
pletely. In this series the apphcations were more than doubled and increased 
to as much as 250 pounds of nitrogen per 2,000,000 pounds of soil, yet no seri- 
ous hindrance was given to nitrogen fixation. The outstanding characteris- 
tic of the data is the lack of variations in nitrogen fixed. The average figures 
for duphcate pots show a fixation of approximately 1200 mgm. for all pots 
except the two receiving 315 mgm. of nitrate nitrogen at the outset, and the 
two receiving 75 mgm. in three successive apphcations. Those pots with 390 
mgm. of added nitrogen gave a fixation of 1187 mgm., corresponding closely 
to the fixation of 1124 and 1212 mgm. in the check pots. The data indicate 
that these amounts of nitrate nitrogen used in this series had no significant 
effect either favorable or unfavorable, and the highest treatment gave no sig- 
nificant variation from the general uniformity of the series. 

In pots 13b and 14b receiving three applications of 25 mgm. each or a total 
equivalent to 50 pounds of nitrogen per 2,000,000 pounds of soil, the fixation 
was the lowest in the series. The crops on these pots were the smallest of 
all pots, as is shown by the weights. The roots were about as heavy in these 
pots as any in the series, but the tops weighed far less than those of any other 
treatments. The same relations held for the total nitrogen in the tops and 
roots of these two pots. That of the tops was less, and that in the roots was 
about the equal of those of the other treatments of the series. 

The fact that the fixation in pots 13b and 14b was not equal to that of 3b 
and 4b might lead one to believe that the application of 50 pounds of nitrate 
at the time the seed was planted gave more fixation than this same amount of 
nitrogen distributed over three applications during the plants' growth. The 
soluble nitrogen applied early may have enabled the plant to estabhsh itself 
better at the outset and fix more nitrogen than when the same amount of 
soluble nitrogen was put into the soil at intervals. Pots 3b and 4b were treated 
exactly the same as pots 13b and 14b, save that the latter two did not receive 
all the nitrogen at the beginning, and such conclusions might be drawn from 
the comparison alone. When both these treatments are compared with the 
check, however, the interpretation must be different. The fixation when all 
the nitrates were applied at the outset was no larger than that of the check 
receiving no treatment, and was less than that of the check when applied 
at intervals during the plants' growth. On this basis the appHcation of all 
nitrogen at the beginning showed no influence on the fixation, while the 
application at intervals kept it down . . 

The series with nitrates gives large amounts of nitrogen fixation for all pots, 
but little variation caused by treatments. It would indicate that nitrates 
used in amounts as high as 1500 pounds per acre do not exert any effect, un- 
less perhaps when the applications are made at intervals during the plants' 
growth rather than at the beginning. 



SYMBIOTIC NITROGEN FIXATION 307 

The second set of pots, whose total nitrogen content of the soil increased by 
approximately 1000 pounds per acre, showed results quite different from those 
it gave in its previous crop of cowpeas. Instead of losses of nitrogen, there 
were marked gains in all pots, indicating that the decaying organic matter 
had no longer lost nitrogen and that the nitrogen taken from the air appeared 
as increase in the total determinations at the close of the series. Check pots 
3 and 4 whose nitrogen in the soil and crop was lower than that in check pots 1 
and 2 in the previous crop of cowpeas, showed similar results again. The 
differences in the soil nitrogen of these two sets of checks amounted to more 
than 100 mgm. and in the crop they were even greater, thus giving a difference 
of over 250 mgm. in the amounts of nitrogen fixed by the two sets of checks. 
The use of one or the other set of checks as a basis for comparison will influence 
the interpretation of the results of the treatments, but only in degree rather 
than in kind, since all the treatments gave results above even the highest of the 
checks. The results from the four check pots were used as a basis in calculat- 
ing the increase, or nitrogen fixation, as influenced by treatment. 

By using the average of the four check pots as a basis for comparison all the 
treatments with organic matter increased the fixation of nitrogen. For those 
pots receiving the heaviest applications the increase is not as large as in the 
others, but all pots below this addition of 5000 pounds of nitrogen per acre 
gave a significant increase in fixation over even the highest of the check pots. 
This indicates that the addition of the organic matter increased the fixation, 
rather than hindered it, and that the sofls richer in total nitrogen permitted the 
plant to draw on the atmospheric supply of this element as well as on that in 
soil. These facts are not in accord with the general statements often made^ 
that the plant fails to draw on the nitrogen of the atmosphere until forced to 
do so by the deficiency in the soil. Here the results were the very reverse and 
the addition of organic nitrogen enabled the plants to take more nitrogen 
from the air as a result of such treatment. Such benefit was suggested even 
in the two previous crops, where the addition of 1000 pounds of nitrogen as 
organic matter gave in every case greater fixation than in soils not so treated. 
According to these data 1000 pounds of nitrogen added as organic matter in- 
creased nitrogen fixation as much as the larger applications used in the series 
and the fixation decreased slightly with increased additions, but this decrease 
was so small as to be insignificant. Even if this decrease were larger there 
would be no cause for alarm since the application of 1000 pounds of nitrogen 
as organic matter is equivalent to approximately 25 tons of clover tops and is■^ 
far greater than is ever applied to soils for fertilizer uses, and such amounts of" 
readily decomposable nitrogenous materials seldom or never occur in common, 
soils. 

The graphic representation of the data in figure 3 shows that cowpeas grow-- 
ing on soils treated with nitrates, or rich in organic matter may utilize atmos-- 
pheric nitrogen even when the amounts of these forms of nitrogen are very- 
large. They show further, that neither the nitrate treatment nor the soil's- 

SOIL SCIENCE, VOL. IX, NO. 5 



308 



WILLIAM ALBERT ALBRECHT 



high total nitrogen content affect nitrogen fixation detrimentally, but rather, 
that the treatment with organic matter favors the process by increasing the 
amount of atmospheric nitrogen taken by this legume. 

The total nitrogen content in the soil of the one series was equivalent to 
775, 1500, 2000, 2440, 2800 and 3240 pounds in 2,000,000 pounds, while the 



1400 



g 

bo 




1000 



600 



i-i 600 



400 



SCO 



&) 



1030 



100 



1£0 



Fixation in ^oils trbated wijbh 
I soils tr- 



nltriites 



Pixa'; 
n1 OTi ir 



Pound 
so 



i]s 



80C0 



^;«a4- 



ion in 

t.npg- 



8 nit; 
treated 



;ro^ en 



2C0 25C» 
Pounds nitrogen adde<f per 
as nitrste. 



sated wi Ux 



per 
with 



3000 



ere in 
lover t 



)p8, 



Fig. 3. Graphs Showing Nitrogen Fixation by Cowpeas on Soils Treated 
WITH Nitrates, and with Clover Tops 

fixations for these were 1005, 1292, 1295, 1261, 1215 and 1155 mgm. per pot, 
respectively. The increases in fixation caused by the organic matter over that 
of the check are 287, 290, 256, 210 and 150 mgm. with the increasing nitrogen 
content of the soil. Accordingly, the soils richer in nitrogen gave somewhat 



SYMBIOTIC NITROGEN FIXATION 309 

less fixation than the soils poorer in this respect, but these differences are not 
significant when compared with the total nitrogen fixed. It may be possible 
that the higher treatments still lost some nitrogen from the soil during this 
time, and if proper corrections could be made they might show the same fixa- 
tion throughout the series. 

Nitrogen changes in soils during the growth of legumes 

Since the soils used in this experiment were carefully analyzed for each crop 
grown on them, they offer an excellent opportunity for study of the changes 
in the nitrogen taking place within the soils themselves. 

The soils in pots 1 to 4 and 1 1 to 20 were used during the entire series of three 
successive crops. The roots were removed as completely as possible to pre- 
vent addition of organic matter in this form. As these soils received applica- 
tions of larger amounts of clover tops at the outset, they offer an interesting 
study of the changes in nitrogen content while this organic matter was decay- 
ing, and while three successive crops of legumes were growing. The following 
data are tabulated to give the changes in the nitrogen content of these soils as 
shown at the close of each crop. In the treated soils, from .which there were 
losses of nitrogen, some estimate of these losses can be made; while in the un- 
treated soil some measure may be taken of the influence of the legume plant on 
the nitrogen content of the soil itself. The figures were obtained from the 
analyses of the soil and organic matter at the beginning and from that of the 
soil alone at the close of each crop series. For the sake of uniformity the data 
are tabulated as pounds per acre of 2,000,000 pounds of soil. The data are 
given in table 17. 

For the soils treated with organic matter, the losses rather than gains in 
nitrogen are the chief characteristics. During the growth of the soybeans no 
losses took place in the soils treated with less than 2890 pounds of nitrogen 
per acre. For this appHcation the average loss was 242 pounds. For the two 
higher treatments the average losses were 382 and 510 pounds. 

During the growth of the second crop, cowpeas, there were losses in all pots, 
and all were greater than occurred in these same soils during the first crop. 
The lowest two treatments, which had lost no nitrogen previously, showed 
decreases equivalent to 205 and 614 pounds per acre, while in those pots with 
larger applications of organic matter, the nitrogen losses were 892, 1254 and 
1539 pounds. In terms of the original amounts of nitrogen applied these 
losses correspond to 20.6, 30.3, 33.0, and 33.0 per cent, respectively. Evi- 
dently the most rapid decomposition and heaviest losses did not take place 
until the time of the second-crop growth, which closed 201 days after the soils 
were treated. Drying and grinding the soil between the two crops may have 
hastened decomposition. 

For the period of time in which the third crop was grown the data show re- 
sults different from those for the_^two preceding periods.^; The treated soils no 



310 



WILLIAM ALBERT ALBRECHT 



longer gave losses of nitrogen, but in all cases gave gains for this element. 
The largest average gain was 77 pounds in the pots receiving 995 pounds of 
nitrogen per acre, but in the other pots the gains decreased with increased 
treatments, though in no regular order. These increases in the soil nitrogen 
are not large, but big enough to be considered as increases as measured by 
accurate analytical determinations varying less than 0.2 mgm. of nitrogen 
in 10-gm. samples of soil in triplicate determinations. This range in difference 
corresponds to 40 pounds per acre, and the error becomes less when three 
samples are taken. Even though these increases were not considered, there 
is no small importance in the fact that these soils produced such heavy crops 

TABLE 17 

Changes during the growth of three crops of legumes in a soil treated with organic matter 

All figures given in pounds per 2,000,000 pounds of soil 





CLOVER TOPS 
ADDED 


NITROGEN 
ADDED 


NITROGEN IN THE SOIL 


POT 


At the 
beginning 


After first 

crop 
(soybeans) 


Increase 


After sec- 
ond crop 
(cowpeas) 


Increase 


After third 

crop 
(cowpeas) 


Increase 


1 

2 
3 

4 

11 
12 
13 
14 
15 
16 
17 
18 
19 
20 


lbs. 
None 
None 
None 
None 

37,766 

37,766 

75,532 

75,532 

113,298 

113,298 

151,064 

151,064 

188,830 

188,830 


lbs. 

None 
None 

None 
None 

995 
995 
1960 
1960 
2890 
2890 
3790 
3790 
4660 
4660 


lbs. 
625 
625 
625 
625 

1620 
1620 

2585 
2585 
3515 
3515 
4415 
4415 
5285 
5285 


lbs. 
5831 

630/ 
6331 
613/ 

16001 
1663/ 

25501 
2615/ 
31851 
3360 J 
4110l 
3955/ 
47701 
4780/ 


lbs. 

-18 

-2 

11 

-2 
-242 
-382 
-510 


lbs. 
7261 
713/ 

6781 
664/ 

14021 
1450/ 
19671 
1968/ 
23651 
2395/ 
27951 
2762/ 
32031 
3269/ 


lbs. 

113 

48 

-205 

-615 

-892 

-1254 

-1538 


lbs. 
8471 
822/ 

7781 
760/ 

14881 
1518/ 
20021 
2006/ 
24481 
2432/ 
28901 
2742/ 
3250\ 
3230/ 


lbs. 

115 

98 

77 
35 
60 
37 
4 



of legumes and yet suffered no loss in nitrogen. In some cases the nitrogen in 
the total crop was over half as much as that originally in the soil. Had the 
crop taken its nitrogen from the soil, such amounts removed could easily have 
been measured by ordinary analytical methods. 

The greatest increases in nitrogen of the soil occurred in the four check pots 
which had been left untreated. During the growth of the soybeans these soils 
showed no significant change, save a very slight loss. During the next crop, 
which was cowpeas, they showed decided gains in nitrogen, and again gave 
similar results for the last crop of this same legume. The soils in all four pots 
increased in nitrogen by growing the cowpeas. It cannot be specifically said 
whether this was due to a direct change in the original soil material itself, or 
to the addition of small roots and nodules that remained in spite of the careful 



SYMBIOTIC NITROGEN FIXATION 311 

attempt to remove them; but it seems most probable that the latter reason was 
sufficient to give the increases recorded. With such gains in soil nitrogen when 
the roots are removed, surely the incorporation of the roots themselves would 
give a decided increase. It has been said for some legumes (25, p. 218) that 
the addition of merely the roots does not increase the nitrogen content of the 
soil, but the above data indicate that the 625 pounds of nitrogen originally 
present in this soil increased when the roots were removed as carefully as pos- 
sible, and would have increased much more if the roots had been included. 
The same may be said of the richer soils during the last crop of cowpeas, all of 
which showed some gains in nitrogen content when the roots were removed. 
Had the roots been left in the soil its nitrogen content would have been raised 
decidedly, even where the total nitrogen of the soil was already equal to 3200 
pounds per acre. None of the soils in the last crop series, whether high or low 
in nitrogen, lost in this element as the result of the removal of the entire crop of 
roots and tops of the plants. All would have been enriched in this respect 
had only the roots been added. 

Distribution of nitrogen in roots and tops of cowpeas 

It has been known that the portions of the total plant substance which are 
in the tops and in the roots are not constant for the different kinds of plants, 
nor are they always constant for the same kind of plant (24, 40, 49) . Similar 
statements may be made concerning the distribution of the constituent ele- 
ments of the plant tissue, especially the nitrogen. The data of this experiment 
were taken in the hope of studying the distribution of the nitrogen in the tops 
and roots, as well as the weight of these plant parts as influenced by the nitro- 
gen content of the soil. The plants were harvested by cutting the stems just 
level with the soil surface, and considering as tops that part above the soil 
level, and as roots all parts below it. Only the two cowpea crops were treated 
in this manner and the data are given for this legume grown in a soil with 
varying amounts of nitrates and in a soil containing varying amounts of or- 
ganic nitrogen. The data were taken from the previous table and are given 
as averages of the dupUcate pots for each treatment (tables 18 and 19). The 
distributions of the weights and nitrogen in the tops and roots are expressed 
as per cent of the total. 

According to the data, the addition of nitrate nitrogen tends to increase the 
weight of the plant top faster than that of the roots. In the first cowpea crop, 
the percentage of the total plant weight in the tops increased gradually while 
that in the roots decreased as the amount of nitrate nitrogen added became 
larger. In the second crop there was also an increase, but not such a marked 
one. The entire second series showed a higher percentage of the total plant 
weight in the top than was shown in the previous series. Evidently some 
other factor was coupled with the nitrogen in the soil to influence the distribu- 
tion of the weights in tops and roots. This may well be expected since the 



312 



WILLIAM ALBERT ALBRECHT 



TABLE 18 

Distribution of nitrogen in the roots and tops of cowpeas 
Soil treated with nitrates 



NITROGEN ADDED 
PER POT 



PART OF PLANT 



PER CENT OF 
TOTAL 



TOTAL NITROGEN 



PER CENT OF 
TOTAL 



First crop 



mgni. 




gm. 




mgm. 




None 


Tops 


15.25 


62.51 


416 


72.40 




Roots 


9.145 


37.48 


158 


27.52 


16.5 


Tops 


15.60 


66.63 


373 


71.45 




Roots 


7.810 


33.36 


149 


28.54 


41.0 


Tops 


16.05 


68.26 


368 


68.91 




Roots 


7.463 


31.73 


166 


31.08 


82.0 


Tops 


16.40 


67.09 


424 


71.99 




Roots 


8.043 


32.90 


165 


28.00 


157.0 


Tops 


15.125 


69.86 


366 


73.34 




Roots 


6.704 


30.71 


131 


26.25 



Second crop 



None 


Tops 


34.32 


75.75 


843 


84.04 




Roots 


10.985 


24.24 


160 


15.95 


75.0 


Tops 


33.04 


78.85 


841 


85.20 




Roots 


8.861 


21.14 


146 


14.79 


150.0 


Tops 


37.55 


75.09 


968 


84.54 




Roots 


12.456 


24.90 


177 


15.45 


225.0 


Tops 


37.20 


76.95 


984 


84.53 




Roots 


11.139 


23.04 


180 


15.46 


315.0 


Tops 


36.93 


76.27 


948 


83.81 




Roots 


11.485 


23.72 


183 


16.18 


390.0 


Tops 


37.90 


79.61 


1134 


86.43 




Roots 


9.705 


20.38 


178 


13.56 


75.0 


Tops 


25.12 


69.27 


605 


78.46 




Roots 


11.139 


30.72 


166 


21.53 



SYMBIOTIC NITROGEN FIXATION 



313 



TABLE 19 

Distribution of nitrogen in the roots and tops of cowpeas 

Soil treated with clover tops 



NITROGEN IN SOIL PART OF PLANT 



PER CENT or 
TOTAL 



TOTAL NITROGEN 



PER CENT OF 
TOTAL 



First crop 



Ibs.^ 




gm. 




mgm. 




None 


Tops 


20.67 


76.05 


581 


81.71 




Roots 


6.508 


23.94 


130 


18.28 


None 


Tops 


20.32 


70.07 


503 


75.07 




Roots 


8.676 


29.92 


167 


24.92 


995 


Tops 


34.92 


76.68 


1049 


83.78 




Roots 


10.615 


23.31 


203 


16.21 


1960 


Tops 


36.37 


75.77 


1051 


84.21 




Roots 


11.630 


24.22 


197 


15.78 


2890 


Tops 


37.82 


75.73 


1170 


84.47 




Roots 


12.114 


24.26 


215 


15.52 


3790 


Tops 


40.45 


81.53 


1273 


88.15 




Roots 


9.161 


18.46 


171 


11.84 


4660 


Tops 


43.57 


82.51 


1490 


91.46 




Roots 


9.235 


17.48 


139 


8 53 







Second crop 






719 


Tops 


33.49 


79.06 


855 


84.90 




Roots 


8.866 


20.91 


152 


15.09 


621 


Tops 


26.10 


74.88 


613 


79.81 




Roots 


8.755 


25.11 


155 


20.18 


1426 


Tops 


37.77 


85.96 


1082 


88.98 




Roots 


6.164 


14.03 


134 


11.01 


1967 


Tops 


37.97 


84.33 


1142 


89.56 




Roots 


7.053 


15.66 


133 


10.43 


2381 


Tops 


35.43 


85.01 


1095 


90.42 




Roots 


6.247 


14.98 


116 


9.57 


2778 


Tops 


37.00 


83.00 


1071 


89.62 




Roots 


7.574 


16.99 


124 


10.37 


3236 


Tops 


35.94 


79.89 


1031 


87.29 




Roots 


9.043 


20.18 


150 


12.70 



^ Pounds in 2,000,000 pounds of soil. 



314 WILLIAM ALBERT ALBRECHT 

crops were grown at different seasons. Nevertheless, the nitrogen in this 
readily soluble form was of influence, and increased the plant growth above the 
ground more than it did the development of the root system. 

In the soils with varying amounts of organic nitrogen, the higher nitrogen 
content of the soil gave a greater portion of the plant weight in the tops, but 
there was no regular increase in this proportion with the regular increase in the 
soil nitrogen. This failed to show the uniformity of increase shown by the 
soils treated with nitrates, but yet indicated that as the soils were richer in 
nitrogen, the weight of the plant above ground increased faster than the 
weight of the roots. 

Considering the distribution of the total nitrogen of the plant, the effects of 
the nitrates of the soil toward increasing the portion of this in the tops are not 
as marked as for increasing the weight of the tops. The data of both series 
fail to show any regular influence, by the nitrates, on the percentage of the total 
plant nitrogen found in the roots or the tops. The effect of the organic nitro- 
gen in the soil was shown as a more rapid increase in the nitrogen of the tops, 
than that of the roots. In the first crop this effect was very decided, giving 10 
per cent more of the plant's total nitrogen in the tops for the soil with high 
nitrogen content than for the soil to which no organic nitrogen had been added. 
In the second crop, all the soils richer in nitrogen than the untreated soil had 
a bigger portion of the plant's nitrogen in the tops, but there was no close cor- 
relation between the soil treatment and the disturbance in the distribution of 
the total nitrogen in the plant tops and roots. Nevertheless, it is evident that 
the nitrogen in the part of the plant above ground increased with the treatment 
of both nitrates and organic matter. 

Between the distribution of the nitrogen, and the fixation of this element, 
there is no correlation in this experiment. It cannot be said that the fixation 
is greater with increased production of that part above the ground, which is 
the relation that one might expect according to Peterman (41). Scarcely 
enough data were secured to warrant a specific statement on this question of 
relation of crop size to the amount of nitrogen fixation. 

IV. SUMMARY 

The experiment herein reported was a study of nitrogen fixation as influenced 
by the nitrogen content of the soil. Variations in the nitrogen of the soil 
were brought about by adding sodium nitrate, and by incorporating organic 
matter in the form of clover tops in a soil containing only 625 pounds of nitro- 
gen per 2,000,000 pounds of soil. This gave variations in mineral nitrogen 
and organic nitrogen, respectively. 

One crop of soybeans and two crops of cowpeas were grown. Some of the 
original soil was used each time for the treatment with nitrates, but the same 
soils were used throughout the three crops for the treatment with organic 
matter. 



SYMBIOTIC NITROGEN FIXATION 315 

Nitrogen fixation is represented as the increase in the total nitrogen in the 
soil and crop at the close of the experiment, over that present in the soil and 
seed at the beginning. 

The deca}dng organic matter occasioned some heavy losses in nitrogen from 
the soil. These losses reached the maximum during the second crop, and had 
apparently disappeared after 200 days. During the second crop, the highest 
losses of nitrogen from the soil were equivalent to 1538 pounds per 2,000,000 
pounds of soil where the original application of clover tops was equivalent to 
4660 pounds of nitrogen per acre. These losses from the soil mth the hea^7• 
applications of organic matter made it impossible in a few instances to measure 
the nitrogen fixation. In those soils, however, to which were added nitrates 
and smaller quantities of organic matter, nitrogen fixation was easily measured 
and a decided amount was found. 

The addition of the approximate eqmvalent of 1000 and 2000 pounds of 
nitrogen as organic matter per acre, corresponding to 18 and 36 tons of clover 
tops, respectively, gave nitrogen fixation for all three crops grown. For the 
last crop grown, during which time the soils no longer showed losses of nitro- 
gen, there were decided gains, or nitrogen fixation, for all treatments with 
organic matter. 

Nitrates did not prohibit nitrogen fixation. Indications in one series of 
cowpeas suggested that increasing amounts of nitrogen applied as sodium 
nitrate lessened the amount of fixation. A later series of the same crop mth 
higher and wider ranges of the appHcation of nitrates failed to show the same 
results, even when as much as 250 pounds of nitrogen was appHed in this 
form. This corresponded to an appHcation of more than 1500 pounds of 
sodiiun nitrate per acre. 

The addition of sodium nitrate to the soil caused the plants to grow better 
at the beginning, but gave no increased nitrogen fixation for two series of cow- 
peas, save in one treatment of 150 pounds of nitrogen per acre, where the fixa- 
tion was slightly greater than that of the check. Fixation in all other treat- 
ments with nitrates was less than that of the check. 

After the soils treated with organic matter ceased to lose nitrogen by decom- 
position, they showed a large nitrogen fixation. The organic matter added 
caused this fixation to be larger than that in the soUs not so treated. The 
increase in the nitrogen fixed as caused by the organic matter, was not propor- 
tional, however, to the amount of organic matter apphed. 

There were some variations in the amounts of nitrogen taken from the air 
by five plants of co^s^eas in each pot. The maximum average fixation for 
duplicate pots was 1295 mgm. on a soU containing the equivalent of 2000 
pounds of total nitrogen per acre. Amounts almost as large were obtained 
in soils treated with 150 pounds of nitrate nitrogen per acre. 

Nodule production was not suppressed to an appreciable extent by any of 
the treatments of nitrate or organic matter. 



316 WILLIAM ALBERT ALBRECHt 

The untreated soils which grew legumes for three successive crops showed 
gains in their nitrogen content as a result of these crop growths, even though 
all roots were removed from the soil as completely as possible. In the last 
crop of cowpeas grown, all soils from which the roots had been removed, be- 
came richer in nitrogen under this treatment, even though some of these soils 
contained more than 3000 pounds of nitrogen per acre before the crop was 
grown, and the crop itself contained more than one-fourth of this amount. 
This fact indicates that the growth of a legume may be large, but yet leave the 
nitrogen content of the soil nearly constant or even increase it, and that the 
addition of the plant roots will increase the soil's nitrogen content when there 
is no loss by leaching. 

As the soil's content of total nitrogen or of nitrate nitrogen was higher, the 
yield of plant tissue became greater; and the greater part of this increase was 
in the tops of the plant rather than in the roots. With a higher nitrogen con- 
tent of the soil, a bigger share of the plant's total nitrogen was also in the tops. 

V. CONCLUSIONS 

1. The results of this study indicate that nitrogen fixation will take place 
in a soil containing large amounts of nitrogen in the form of either nitrates or 
organic matter. 

2. No injurious effects on nitrogen fixation were caused by nitrates in this 
experiment, and if such ever occur under conditions similar to those which 
obtained in this study, the apphcation of nitrates must be many times larger 
than is ever applied in agricultural practice. 

3. Nodules are produced when large amounts of organic nitrogen are present 
in the soil, and good legume growth results even when sufficient organic matter 
is present to give large losses of volatile nitrogen from the soils. 

4. The addition of some organic matter may increase the amount of nitro- 
gen fixed by cowpeas. 

5. In soils containing varying amounts of total nitrogen, as much fixation 
of nitrogen by cowpeas may be expected in one with 3000 pounds of total 
nitrogen, as in one with lesser amounts. According to the data given, varia- 
tions in the amount of total nitrogen in a soil failed to exert any varying influ- 
ence on the amount of nitrogen fixed. 

ACKNOWLEDGEMENT 

This opportunity is taken by the author to express his obligations to Pro- 
fessor A. L. Whiting, of the Division of Soil Biology, under whose able direc- 
tion this study was conducted. 



SYMBIOTIC NITROGEN FIXATION 317 

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(3) Baessler, p. 1893 Untersuchungen uber verschiedene Griindungspflanzen und 

die Wirkung derselben auf den Ertrag der Nachfruchte. In Wehmschr. Pommer. 
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(4) Beijerinck, M. W. 1891 Over ophooping van atmospharische Stickstoff in Cul- 

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(7) Btirrill, T. J., AND Hansen, R. 1917 Is sjonbiosis possible between legume 

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(8) Conn, H. W. 1909 Agricultural Bacteriology, p. 147-148. Blakiston, Philadelphia. 

(9) Devarda, a. 1894 Uber die direkte Bestimming des Sticks toff es im Saltpeter. 

In Ztschr. Anal. Chem., Bd. 33, p. 113. 

(10) De Vries, H. 1877 Beitrage zur specialle Physiologie landwirtschaftlichen Kultur- 

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(11) EwART, A. J. 1915 The influence of nitrates on the development of root tubercles. 

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(13) Frank, B. 1889 Uber die Pilzsyrabiose der Leguminosen. In Ber. Deut. Bot. 

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(14) Frank, B. 1892 Die Assimilation der freien Stickstoflfe bei den Pflanzen in ihrer 

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(19) Hellriegel, H., and Wilfarth, H. 1888 Untersuchungen ueber die Stickstoff- 

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(20) Hercke, a. 1912 On the appropriation of nitrogen by legumes. In Kieserlet 

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318 WILLIAM ALBERT ALBRECHT 

(21) Hercke, S. 1913 Contributions on nitrogen fixation and nutrition of Bacillus 

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SYMBIOTIC NITROGEN FIXATION 319 

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PLATE 1 

Soybeans on Soil Treated with Nitrates, Clover Tops, and with Clover 
Tops and Nitrates 



320 



SYMBIOTIC NITROGEN FIXATION 

WILLIAM ALBERT ALBRECHT 



PLATE 1 





C<3-A 







1 




^1% • 


f 


1 : 


1 


H- 




^- 1 n> N __^_-N<,N;]^«b ItmlM 



321 



PLATE 2 



•CowPEAS, AT 50 Days, on Soil Treated with Nitrates (Above) and Cowpeas 
AS THE Second Crop on Soil Treated with Clover Tops (Below) 



322 



SYMBIOTIC XTTROGEX FTX.\TIOX 

WILIIAII ALBERT AISSXCHT 



PLATE 2 



'%S^ ^f<^ "^^^ **T>^ ^ 





son. scEzxci:, tol. ix, xo. o 



PLATE 3 

CowPEAS, AT 75 Days, on Soil Treated with Nitrates (x^bove) and Cowpeas 
AS the Second Crop on Soil Treated with Clover Tops (Below) 



324 



SYMBIOTIC NITROGEN FIXATION 

WILLIAM ALBERT ALBRECHT 



PLATE 3, 





"* Tlfli^' 





325 



PLATE 4 

CowPEAS ON Soil Treated with Nitrates (Above) and Cowpeas as the 
Third Crop on Soil Treated with Clox^er Tops (Below) 



326 



SYMBIOTIC NITROGEN FIXATION 

WILLIAM ALBERT ALBRECHT 



PLATE 4 





327 



BIOGRAPHICAL SKETCH 

William Albert Albrecht was born on a farm near Flanagan, Livingston 
County, Illinois, September 12, 1888. He secured his common-school educa- 
tion in the district school, and his preparatory work in the academy of Bluff ton 
College, Bluffton, Ohio. In the fall of 1907 he entered the University of 
Illinois, College of Liberal Arts and Sciences, from which he received the degree 
of Bachelor of Arts in 1911. The following year was spent as teacher of Latin 
in Bluffton College, Bluffton, Ohio, after which he returned to the University 
of Illinois to enter the College of Agriculture, where he received the degree of 
Bachelor of Science in 1914. During that year he was granted a scholarship 
and received the degree of Master of Science. In 1915 he was a fellow in 
agronomy in the University of Illinois, enabling him to pursue graduate work 
during that time. In the fall of 1916 he went to the University of Missouri, 
College of Agriculture, where he is now associate professor in soils. 

He is author of the following papers: "Changes in the Nitrogen Content 
of Stored Soils," published in the Journal of the American Society of Agronomy, 
Vol. 10, February, 1918, and "Soil Inoculation for Legumes," accepted for 
publication as Missouri Agricultural Experiment Station Circular 86. 

He is a member of the following societies: Gamma Alpha, Alpha Zeta, 
Sigma Xi, American Society of Agronomy, and Society of American Bacteriolo- 
gists. 



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